Biological fertilizer based on yeasts

ABSTRACT

The present invention provides biological fertilizer compositions that comprise yeast cells that have an enhanced ability to fix atmospheric nitrogen, decompose phosphorus minerals and compounds, decompose potassium minerals and compounds, decompose complex carbon compounds, over produce growth factors, and over produce ATP. The biological fertilizer composition of the invention can replace mineral fertilizers in supplying nitrogen, phosphorus, and potassium to crop plants. Methods of manufacturing the biological fertilizer compositions and methods of uses are also encompassed.

1. FIELD OF THE INVENTION

The invention relates to a biological fertilizer that comprises yeastsfor fixing atmospheric nitrogen, and decomposing insoluble compoundscontaining phosphorus, potassium and/or carbon. The invention alsorelates to methods for manufacturing the biological fertilizer, andmethods for using the biological fertilizer to increase crop yields.

2. BACKGROUND OF THE INVENTION

Use of fertilizer is essential in supporting the growth of high yieldcrops. Of the basic nutrients that plants need for healthy growth, largeamounts of nitrogen (taken up as NO₃ ⁻ or NH₄ ⁺), phosphorus (taken upas H₂PO₄ ⁻), and potassium (taken up as K⁺) nutrients are required bymost crops on most soils (Wichmann, W., et al., IFA World Fertilizer UseManual). Such large amounts of nitrogen, phosphorus, and potassiumnutrients are supplied mainly in the form of mineral fertilizers, eitherprocessed natural minerals or manufactured chemicals (K. F. Isherwood,1998, Mineral Fertilizer Use and the Environment, United NationsEnvironmental Programme Technical Report No. 26.). The development anduse of mineral fertilizers since the 1940s has permitted significantincreases in crop yields on the same to slightly less amount of croplandto support today's enormous population. Without such advances inagriculture, a great amount of pastures and forests would have beenconverted into cropland. (K. F. Isherwood, 1998, Mineral Fertilizer Useand the Environment, United Nations Environmental Programme TechnicalReport No. 26.)

Despite the importance of mineral fertilizers in providing mankind withabundant agricultural products, the harm done to the environment hasbeen recognized in the recent years. Mineral fertilizers may incurreddamages to soils. For example, most nitrogen fertilizers may acidifysoils, thereby adversely affecting the growth of plants and other soilorganisms. Extensive use of chemical nitrogen fertilizers may alsoinhibit the activity of natural nitrogen fixing microorganisms, therebydecreasing the natural fertility of soils. Mineral fertilizers may alsointroduce toxic substances into soil and produce. For example, phosphatefertilizers processed from rock phosphate often contain small amounts oftoxic elements, such as cadmium, which may build up in soil and be takenup by plants. The long term use of mineral fertilizers may also causesevere environmental pollution. For example, the loss of nitrogen andphosphate fertilizers due to leaching and soil erosion has led tocontamination of soil and ground water, and eutrophication of surfacewater. Cleaning up polluted soil and water has been a complicated anddifficult task. The cost for such a task is also astronomical.

In search for a solution to the problem, some are going back to organicfertilizers. As is well known, organic fertilizers come from manydifferent sources. Types of organic fertilizer include farm wastes, suchas crop residues and animal manures; residues from plant and animalproducts, such as wood materials; and town wastes, such as sewage(Wichmann, W., et al., IFA World Fertilizer Use Manual). Organicfertilizers are usually low in nutrients and less effective insupporting plant growth. For example, the total nutrients in cattlemanure is less than 2%, and the nitrogen nutrients therein are moredifficult to be effectively utilized due to their losses into theenvironment (K. F. Isherwood, 1998. Mineral Fertilizer Use and theEnvironment, United Nations Environmental Programme Technical Report No.26.). Normally, very large amount of organic fertilizers have to beapplied to soil. To reach high crop yield, organic fertilizers have onlybeen used to supplement mineral fertilizers. Therefore, the problemswith mineral fertilizers cannot be satisfactorily solved by substitutingmineral fertilizer with organic fertilizer. Furthermore, organicfertilizers also have created environmental problems. For example, someorganic fertilizers, if unprocessed, contains pathogenic microorganisms,such as E. coli, Salmonella, and Coccidae. Organic fertilizers may alsocontain toxic chemicals and may produce undesirable odor. The use oforganic fertilizer also contribute to the contamination andeutrophication of the natural water system. Therefore, in many parts ofthe world, including the United States, laws and regulations have beenestablished imposing considerable restriction on both the compositionand the usage of organic fertilizers.

Biological fertilizers utilizing microorganisms have been proposed asalternatives to mineral fertilizers. Naturally occurring nitrogen fixingmicroorganisms including bacteria, such as Rhizobium, Azotobacter, andAzospirillum, (See for example, U.S. Pat. No. 5,071,462) and fungi, suchas Aspergillus flavus-oryzae, (See, for example, U.S. Pat. No.4,670,037) have been utilized in biological fertilizers. Naturallyoccurring microorganisms capable of solubilizing rock phosphate ore orother insoluble phosphates into soluble phosphates have also beenutilized in biological fertilizers either separately (e.g., U.S. Pat.No. 5,912,398) or in combination with nitrogen fixing microorganisms(e.g., U.S. Pat. No. 5,484,464). Genetically modified bacterial strainshave also been developed and utilized in biological fertilizers. Anapproach based on recombinant DNA techniques has been developed tocreate more effective nitrogen fixing, phosphorus decomposing, andpotassium decomposing bacterial strains for use in a biologicalfertilizer, see, for example, U.S. Pat. No. 5,578,486; PCT publicationWO 95/09814; Chinese patent publication: CN 1081662A; CN 1082016A; CN1082017A; CN 1103060A; and CN 1109595A.

However, the biological fertilizers that are based on naturallyoccurring microorganisms are generally not efficient enough toeffectively replace mineral fertilizers. It is therefore important todevelop biological fertilizers that can replace mineral fertilizers insupplying nitrogen, phosphorus, and potassium to crops for producinghigh quality agricultural products while avoiding the problemsassociated with mineral fertilizers. The present invention provides abiological fertilizer based on yeasts, which can replace mineralfertilizers.

Citation of documents herein is not intended as an admission that any ofthe documents cited herein is pertinent prior art, or an admission thatthe cited documents are considered material to the patentability of theclaims of the present application. All statements as to the date orrepresentations as to the contents of these documents are based on theinformation available to the applicant and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

3. SUMMARY OF THE INVENTION

The present invention relates to biological fertilizers. The biologicalfertilizer compositions of the invention may comprise up to sixdifferent yeast cell components, an organic substrate component and/oran inorganic substrate component. In particular, the yeast cellcomponents of the composition are capable of fixing atmosphericnitrogen, decomposing insoluble minerals or compounds, decomposingcomplex carbon materials or compounds, overproducing growth factors, oroverproducing ATP, respectively.

The present invention uses yeasts that are commercially available and/oraccessible to the public, such as but not limited to Saccharomycescerevisiae. The yeast cell components of the invention are produced byculturing yeast cells under activation conditions such that theabilities of the cells to fix atmospheric nitrogen, to decomposeinsoluble phosphorus minerals or compounds, to decompose insolublepotassium minerals or compounds, and to decompose complex carbonmaterials or compounds are activated or enhanced. The yeast cells canalso be cultured under conditions such that their abilities to produceexcess growth factors or ATP are activated or enhanced. Yeast cellsexhibiting such activities are useful in converting nitrogen from theatmosphere to nitrogenous compounds that can be used by plants asnutrients, releasing the otherwise insoluble phosphorus, potassium andcarbon from minerals and complex molecules, such that these elementsbecome available in a form that the plant can utilize for growth. Someyeast cells in the fertilizer are used for supporting other plantnutrient-providing yeast cells by supplying them with growth factors andATP.

The present invention also involves the use of a wide variety of organicand inorganic materials in the fertilizer to support the growth of theyeast strains of the present invention. In one embodiment, thefertilizer is produced by mixing coal-mine waste and rock phosphate withthe yeast strains. In another embodiment, the fertilizer is produced bymixing animal manures, and optionally, a biological disinfectant, withthe yeast strains. In yet another embodiment, the fertilizer is producedby mixing sludge from sewage water treatment plant and a biologicaldisinfectant with the yeast strains.

The invention also relates to methods for manufacturing the fertilizercomprising mixing, drying, and packing the yeast strains of the presentinvention and the organic and/or inorganic materials.

The invention further relates to methods for using the fertilizer of thepresent invention. The biological fertilizers of the present inventionare used to support and enhance the growth and maturation of a widevariety of plants.

4. BRIEF DESCRIPTION OF FIGURES

FIG. 1. Activation of yeast cells. 1 yeast culture; 2 container; 3electromagnetic field source.

FIG. 2. Formation of symbiosis-like relationships among yeast strains. 4electromagnetic field source for nitrogen-fixing yeast; 5electromagnetic field source for P-decomposing yeast; 6 electromagneticfield source for K-decomposing yeast; 7 electromagnetic field source forC-decomposing yeast; 8 yeast culture; 9 container.

FIG. 3. Adaptation of yeast cells to a soil type. 10 electrode; 11container; 12 electrode; 13 yeast culture; 14 electromagnetic fieldsource; 15 temperature controller.

FIG. 4. Organic material grinding process. 16 organic raw material; 17crusher; 18 grinder; 19 organic material in powder form.

FIG. 5. Inorganic material grinding process. 20 inorganic raw material;21 crusher, 22 grinder, 23 inorganic material in powder form.

FIG. 6. Yeast fermentation process. 24 activated yeast cells; 25 tankfor culturing yeast cells, starch:water (35° C.)=1:2.5, semi-aerobicfermentation at 28 to 30° C. for 48 to 72 hours; 26 harvested culture.

FIG. 7. Mixing organic and inorganic raw materials. 27 inorganicmaterials; 28 starch; 29 organic materials; 30 mixer; 31 mixture; 32mixture to be transported to fertilizer production stage.

FIG. 8. Mixing yeast cells. 33 inlets for nitrogen-fixing,P-decomposing, K-decomposing, and C-decomposing yeasts; 34 mixing tank;35 ATP-producing yeast; 36 GP-producing yeast; 37 mixture of yeasts; 38mixture to be transported to fertilizer production stage.

FIG. 9. Fertilizer production process. 39 mixture of yeast; 40 mixtureof organic and inorganic materials; 41 granulizer; 42 fertilizergranules.

FIG. 10. Drying process. 43 fertilizer granules; 44 first dryer; 45second dryer; 46 dried fertilizer.

FIG. 11. Cooling and packaging process. 47 dried fertilizer; 48 cooler;49 separator; 50 bulk bag filler; 51 final product.

5. DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present-invention provides biological fertilizercompositions that comprise yeast cells. The present invention alsoprovides in various embodiments, methods for manufacturing thebiological fertilizer compositions as well as methods for using thebiological fertilizer compositions.

The biological fertilizer compositions of the invention can replacechemical/mineral fertilizers in supplying nitrogen (N), phosphorus (P),and potassium (K) to plants, especially crop plants. The biologicalfertilizer compositions of the present invention can increase cropyields by 10-60%. Because the biological fertilizers of the presentinvention utilize metabolic activities of living yeasts to convert rawmaterials, such as atmospheric nitrogen and phosphorus and potassiumminerals, into plant nutrients, the conversion and release of suchnutrients by the yeast cells is regulated in part by the nutrientcontent of the soil. The nutrient content of the soil in turn depends inpart on both the environment and the changing needs of plants.Therefore, the release of plant nutrients by the biological fertilizercompositions is adaptable to the soil condition and can be sustainedover a period of time.

In one embodiment, the biological fertilizer compositions of theinvention comprise one or more yeast cell components. A yeast cellcomponent of the biological fertilizer compositions comprises aplurality of yeast cells which are capable of performing one of thefollowing functions, each of which results in the provision of one typeof nutrients to plants: (1) fixation of atmospheric nitrogen; (2)decomposition of phosphorus minerals or compounds; (3) decomposition ofpotassium minerals or compounds; (4) decomposition of complex or highmolecular weight carbon materials or compounds. Additional yeast cellcomponents can be included to produce growth factors and. ATP to supportthe other yeasts in the fertilizer compositions.

The biological fertilizer compositions of the invention can furthercomprises an organic substrate component, and/or an inorganic substratecomponent. The organic substrate component of the fertilizercompositions is a primary carbon source for the yeast cells in thefertilizer. The inorganic substrate component provides the yeast cellsin the fertilizer compositions minerals, materials, and compoundscontaining phosphorus and/or potassium. The organic and inorganicsubstrate component may also provide the plants with other minerals suchas but not limited to calcium, magnesium, and sulfur; andmicronutrients, such as but not limited to boron, copper, iron,manganese, molybdenum, and zinc.

As used herein, the term “nitrogen fixation” or “fixation of atmosphericnitrogen” encompasses biological processes in which molecular nitrogenor nitrogen in the atmosphere is converted into one or more nitrogenous(N) compounds, including but not limited to, ammonia, ammonium salts,urea, and nitrates.

As used herein, the phrase “decomposition of phosphorus minerals orcompounds” refers to biological processes which convert phosphorus (P)compounds, such as but not limited to those water-insoluble phosphoruscompounds present in rock phosphate, into one or more differentphosphorus compound(s) which can be more readily used for survivaland/or growth by plants and other yeasts. For example, the resultingphosphorus compounds may be more soluble in water, and can thus be takenup by the roots of plants.

As used herein, the phrase “decomposition of potassium minerals orcompounds” refers biological processes which convert potassium (K)compounds, such as but not limited to those water-insoluble potassiumcompounds present in potassium mica, into one or more differentpotassium compound(s) which can be more readily used for survival and/orgrowth by plants and other yeasts. For example, the resulting potassiumcompounds may be more soluble in water, and can thus be taken up by theroots of plants.

As used herein, the phrase “decomposition of complex or high molecularweight carbon minerals, materials or compounds” refers to the biologicalconversion of a complex organic or inorganic carbon molecule into one ormore carbon molecule(s) which usually are of a lower molecular weight,and can be more readily used for survival and/or growth by plants andother organisms, including other yeasts. For example, it encompasses theconversion of high molecular weight carbon compounds in weathered coalto simple carbohydrates, such as pentose and hexose. This processincludes those reactions where long chains of carbon atoms in apolymeric carbon compound are cleaved.

As used herein, the term “growth factors” refers to molecules commonlyrequired for growth of yeasts, including but not limited to vitamins, inparticular, vitamin B complexes, e.g., vitamin B-1, riboflavin (vitaminB-2), vitamin B-12, niacin (B-3), pyridoxine (B-6), pantothenic acid(B-5); folic acid; biotin; para-aminobenzoic acid; choline; andinositol.

A wide variety of organic and inorganic materials may be used to supplythe phosphorus, potassium, and complex high molecular weight carbonminerals, materials and compounds to be converted by the yeast cellsinto nutrients for use by the yeasts and the plants. The organic andinorganic materials that may be used in conjunction with the presentinvention include, but not limited to, minerals, such as but not limitedto phosphate rock or rock phosphate, apatite, phosphorite, sylvinite,halite, carnalitite, potassium mica, lignite; industrial materials orwastes, such as but not limited to coal-mine waste, weathered coal,coal-powder, and hydrocarbon waste; environmental materials and wastes,such as but not limited to sludge from sewage water treatment plant andland fills, muds, such as turf mud, mud from river and lake bed; organicwastes, such as but not limited to waste and manure from urban areas andanimal manure, such as poultry manure, cattle manure, hog manure, sheepmanure, and guano, waste materials from plants, waste material fromanimals including fish meal, bone meal, human waste, dried blood, etc.,and products or by-products from fermentation of plant materialscontaining cellulose, starch and/or other carbohydrates.

In addition, depending on needs, a disinfectant may be included in thebiological fertilizer compositions. An environmentally safe disinfectantis preferred. For example, a biological disinfectant, super-CM₆₁ can beused with environmental and organic wastes, such as waste and manurefrom urban areas and animal manure.

In various embodiments, the biological fertilizer compositions of thepresent invention comprises at least one yeast cell component, andpreferably six yeast cell components. The inventor discovered that,under certain culture conditions, various yeast strains can be inducedto exhibit the following six activities: (1) fixation of atmosphericnitrogen; (2) decomposition of phosphorus minerals or compounds; (3)decomposition of potassium minerals or compounds; (4) decomposition ofcomplex or high molecular weight carbon materials or compounds; (5)production of excess growth factors in an amount that is sufficient tosupport the needs of other yeast strains in the fertilizer composition;and (6) production of excess ATP in an amount that is sufficient tosupport the needs of other yeast strains in the fertilizer composition.The culture condition determines the activity which is activated orenhanced in the cultured yeasts. The specific culture conditions foreach of the six activities are described in details in sections 5.1-5.6respectively.

According to the invention, a yeast cell component of the biologicalfertilizer is produced by culturing a plurality of yeast cells in anappropriate culture medium in the presence of an electromagnetic field.The electromagnetic field can be generated by various means well knownin the art. A schematic illustration of an exemplary setup is depictedin FIG. 1. The electromagnetic field of a desired frequency andamplitude is generated by an electromagnetic source (3) which comprisesone or more signal generators that are capable of generatingelectromagnetic waves, preferably sinusoidal waves, in the frequencyrange of 100 MHz-2000 MHz. If desirable, a signal amplifier can also beused to increase the output. The electromagnetic field can be applied tothe culture by a variety of means including placing the culture in closeproximity to the signal emitters. In one embodiment, the electromagneticfield is applied by electrodes that are submerged in the culture (1). Ina preferred embodiment, one of the electrodes is a metal plate, and theother electrode comprises a plurality of wires configured inside thecontainer (2) so that the energy of the electromagnetic field can beevenly distributed in the culture. The number of electrode wires useddepends on both the volume of the culture and the diameter of the wire.In preferred embodiments, for a culture having a volume up to 5000 ml,one electrode wire having a diameter of between 0.1-1.2 mm can be usedfor each 100 ml of culture; for a culture having a volume greater than1000 l, one electrode wire having a diameter of between 3-30 mm can beused for each 1000 l of culture. The types of yeasts contemplated foruse in the invention include without limitation, yeasts of the genera ofSaccharomyces, Schizosaccharomyces, Sporobolomyces, Torulopsis,Trichosporon, Wickerhamia, Ashbya, Blastomyces, Candida, Citeromyces,Crebrothecium, Cryptococcus, Debaryomyces, Endomycopsis; Geotrichum,Hansenula, Kloeckera, Lipomyces, Pichia, Rhodosporidium, andRhodotorula. Non-limiting examples of yeast strains includeSaccharomyces cerevisiae Hansen, ACCC2034, ACCC2035, ACCC2036, ACCC2037,ACCC2038, ACCC2039, ACCC2040, ACCC2041, ACCC2042, AS2.1, AS2.4, AS2.11,AS2.14, AS2.16, AS2.56, AS2.69, AS2.70, AS2.93, AS2.98, AS2.101,AS2.109, AS2.110, AS2.112, AS2.139, AS2.173, AS2.174, AS2.182, AS2.196,AS2.242, AS2.336, AS2.346, AS2.369, AS2.374, AS2.375, AS2.379, AS2.380,AS2.382, AS2.390, AS2.393, AS2.395, AS2.396, AS2.397, AS2.398, AS2.399,AS2.400, AS2.406, AS2.408, AS2.409, AS2.413, AS2.414, AS2.415, AS2.416,AS2.422, AS2.423, AS2.430, AS2.431, AS2.432, AS2.451, AS2.452, AS2.453,AS2.458, AS2.460, AS2.463, AS2.467, AS2.486, AS2.501, AS2.502, AS2.503,AS2.504, AS2.516, AS2.535, AS2.536, AS2.558, AS2.560, AS2.561, AS2.562,AS2.576, AS2.593, AS2.594, AS2.614, AS2.620, AS2.628, AS2.631, AS2.666,AS2.982, AS2.1190, AS2.1364, AS2.1396, IFFI 11001, IFFI 1002, IFFI 1005,IFFI 1006, IFFI 1008, IFFI 1009, IFFI 1010, IFFI 1012, IFFI 1021, IFFI1027, IFFI 1037, IFFI 1042, IFFI 1043, IFFI 10451, IFFI 1048, IFFI 1049,IFFI 1050, IFFI 1052, IFFI 1059, IFFI 1060, IFFI 1063, IFFI 1202, IFFI1203, IFFI 1206, IFFI 1209, IFFI 1210, IFFI 1211, IFFI 1212, IFFI 1213,IFFI 1215, IFFI 1220, IFFI 1221, IFFI 1224, IFFI 1247, IFFI 1251, IFFI1270, IFFI 1277, IFFI 1287, IFFI 1289, IFFI 1290, IFFI 1291, IFFI 1291,IFFI 1292, IFFI 1293, IFFI 1297, IFFI 1300, IFFI 1301, IFFI 1302, IFFI1307, IFFI 1308, IFFI 1309, IFFI 1310, IFFI 1311, IFFI 1331, IFFI 1335,IFFI 1336, IFFI 1337, IFFI 1338, IFFI 1339, IFFI 1340, IFFI 1345, IFFI1348, IFFI 1396, IFFI 1397, IFFI 1399, IFFI 1411, IFFI 1413, ACCC2043,AS2.2, AS2.3, AS2.8, AS2.53, AS2.163, AS2.168, AS2.483, AS2.541,AS2.559, AS2.606, AS2.607, AS2.611, AS2.612; Saccharomyces chevalieriGuillermond, AS2.131, AS2.213; Saccharomyces delbrueckii Lindner,AS2.285; Saccharomyces delbrueckii Lindner ver. mongolicus Lodder,AS2.209, AS2.11157; Saccharomyces exiguus Hansen, AS2.349, AS2.1158;Saccharomyces fermentati (Saito) Lodder et van Rij, AS2.286, AS2.343;Saccharomyces logos van laer et Denamur ex Jorgensen, AS2.156, AS2.327,AS2.335; Saccharomyces mellis Lodder et Kreger Van Rij, AS2.195;Saccharomyces microellipsoides Osterwalder, AS2.699; Saccharomycesoviformis Osterwalder, AS2.100; Saccharomyces rosei Lodder et kreger vanRij, AS2.287; Saccharomyces rouxi Boutroux, AS2.178, AS2.180, AS2370,AS2.371; Saccharomyces sake Yabe, ACCC2045; Saccharomyces uvarum Beyer,IFFI 1023, IFFI 1032, IFFI 1036, IFFI 1044, IFFI 1072, IFFI 1205, IFFI1207; Saccharomyces willianus Saccardo, AS2.5, AS2.7, AS2.119, AS2.152,AS2.293, AS2.381, AS2.392, AS2.434, AS2.614, AS2.1189; Saccharomycessp., AS2.311; Saccharomyces ludwigii Hansen, ACCC2044, AS2.243, AS2.508;Saccharomyces sinenses Yue, AS2.1395; Schizosaccharomyces octosporusBeijerinck, ACCC 2046, AS2.1148; Schizosaccharomyces pombe Linder,ACCC2047, ACCC2048, AS2.248, AS2.249, AS2.255, AS2.257, AS2.259,AS2.260, AS2.274, AS2.994, AS2.1043, AS2.1149, AS2.1178, IFFI.1056;Sporobolomyces roseus Klyver et van Niel, ACCC 2049, ACCC 2050, AS2.619,AS2.962, AS2.1036; Sporobolomyces salmonicolor (Fischer et Brebeck)Kluyver et van Niel, ACCC2051, AS2.261, AS2.262; Torulopsiscandida(Saito)Lodder, ACCC2052, AS2.270; Torulopsis famta(Harrison)Lodder et van Rij, ACCC2053, AS2.685; Torulopsis globosa(Olson et Hammer)Lodder et van Rij, ACCC2054, AS2.202; Torulopsisinconspicua Lodder et van Rij, AS2.75; Trichosporon behrendoo Lodder etKreger van Rij, ACCC2055, AS2.1193; Trichosporon capitatum Diddens etLodder, ACCC2056, AS2.1385; Trichosporon cutaneum(de Beurm et al.)Ota,ACCC2057, AS2.25, AS2.570, AS2.571, AS2.1374; Wickerhamia fluo{overscore(r)}esens (Soneda) Soneda, ACCC2058, AS2.1388;, Ashbya gossypii (Ashbyet Nowell) Guillermond, ACCC2001, AS2.475, AS2.1176; Blastomycesdermatitidis Gilehrist et Stikes, ID(D 10)23; Candida albicans (Robin)Berkhout, ACCC2002, AS2.538, ID 16u(C1)u, ID 61v(C1)v; Candida arborea,AS2.566; Candida guillermondii(Castellani) Langeron et guerra, AS2.63,ID 21 a(C5)a, ID 21 b(C5)b; Candida Krusei (Castellani) Berkhout,AS2.1045; Candida lambica(Lindner et Genoud) van.Uden et Buckley,AS2.1182; Candida lipolytica (Harrison) Diddens et Lodder, AS2.1207,AS2.1216, AS2.1220, AS2.1379, AS2.1398, AS2.1399, AS2.1400; Candidaparakrusei (Castellani et Chalmer) Langeron et Guerra, ID 19 a(C4)a, ID19 b(C4)b, ID 19 c(C4)c, ID 19 d(C4)d; Candida parapsilosis (Ashford)Langeron et Talice, AS2.590; Candida parapsilosis (Ashford) et TaliceVar.imtermedia Van Rij et Verona, AS2.491; Candida pseudotropicalis(Castellani) Basgal, AS2.68, ID64(C3); Candida pulcherrima (Lindner)Windisch, AS2.492; Candida robusta Diddens et Lodder, AS2.1195; Candidarugousa (Anderson) Diddens et Loddeer, AS2.511, AS2.1367, AS2.1369,AS2.1372, AS2.1373, AS2.1377, AS2.1378, AS2.1384; Candida tropicalis(Castellani) Berkout, ACCC2004, ACCC2005, ACCC2006, AS2.164, AS2.402,AS2.564, AS2.565, AS2.567, AS2.568, AS2.617, AS2.637, AS2.1387,AS2.1397, ID 17 a(C₂)a, ID 17 b(C₂)b, ID 17 d(C₂)d; Candida utilisHenneberg Lodder et Kreger Van Rij, AS2.120, AS2.281, AS2.1180;Citeromyces matritensis (Santa Maria) Santa Maria, AS2.1401;Crebrothecium ashbyii (Guillermond) Routein, ACCC2013, ACCC2014,AS2.481, AS2.482, AS2.1197; Cryptococcus laurentii (Kufferath) Skinner,ACCC2007, AS2.114, ID 95 (y₂); Cryptococcus neoformans (Sanfelice)Vuillemin, ID 25 u(D₂)u, ID 25 v(D₂)v, ID 25 w(D₂)w; Debaryomyceshansenii (Zopf) Lodder et Kreger-van Rij, ACCC2010, AS2.45; Debaryomyceskloeckeri Guilliermond et Peju, ACCC2008, ACCC2009, AS2.33, AS2.34,AS2.494; Debaryomyces sp., ACCC2011, ACCC2012; Endomycopsis fibuligera(Lindner) Dekker, ACCC2015, AS2.1145; Eremothecium ashbyii Guilliermond;Geotrichum candidum Link, ACCC2016, AS2.361, AS2.498, AS2.616, AS2.1035,AS2.1062, AS2.1080, AS2.1132, AS2.1175, AS2.1183; Geotrichum ludwigii(Hansen) Fanf et al., AS2.363; Geotrichum robustum Fang et al.,ACCC2017, AS2.621; Geotrichum suaveolens (Krzemecki) Fang et al.,AS2.364; Hansenula anomala (Hansen) H et P sydow, ACCC2018, AS2.294,AS2.295, AS2.296, AS2.297, AS2.298, AS2.299, AS2.300, AS2.302, AS2.338,AS2.339, AS2.340, AS2.341, AS2.470, AS2.592, AS2.641, AS2.642, AS2.735,AS2.782, AS2.794; Hansenula arabitolgens Fang, AS2.887; Hansenulajadinii Wickerham, ACCC2019; Hansenula saturnus (Klocker) H et P sydow,ACCC2020, AS2.303; Hansenula schneggii (Weber) Dekker, AS2.304;Hansenula subpelliculosa Bedford, AS2.740, AS2.760, AS2.761, AS2.770,AS2.783, AS2.790, AS2.798, AS2.866; Kloeckera apiculata (Reess emend.Klocker) Janke, ACCC2021, ACCC2022, ACCC2023, AS2.197, AS2.496, AS2.711,AS2.714; Lipomyces starkeyi Lodder et van Rij, ACCC2024, AS2.1390;Pichia farinosa (Lindner) Hansen, ACCC2025, ACCC2026, AS2.86, AS2.87,AS2.705, AS2.803; Pichia membranaefaciens Hansen, ACCC2027, AS2.89,AS2.661, AS2.1039; Rhodosporidium toruloides Banno, ACCC2028, AS2.1389;Rhodotorula aurantiaca (Saito) Lodder, ACCC2029, AS2.280; Rhodotorulaglutinis (Fresenius) Harrison, ACCC2030, AS2.102, AS2.107, AS2.278,AS2.499, AS2.694, AS2.703, AS2.704, AS2.1146; Rhodotorula minuta (Saito)Harrison, AS2.277; Rhodotorula rubar (Demme) Lodder, ACCC2031, AS2.21,AS2.22, AS2.103, AS2.105, AS2.108, AS2.140, AS2.166, AS2.272, AS2.279,AS2.282; Rhodotorula sinesis Lee, AS2.1391; Saccharomyces bailiiLindner, AS2.312; and Saccharomyces carlsbergensis Hansen, ACCC2032,ACCC2033, AS2.113, AS2.116, AS2.118, AS2.121, AS2.132, AS2.162, AS2.189,AS2.200, AS2.216, AS2.265, AS2.377, AS2.417, AS2.420, AS2.440, AS2.441,AS2.443, AS2.444, AS2.459, AS2.595, AS2.605, AS2.638, AS2.742, AS2.745,AS2.748, AS2.1042.

Certain yeast species that can be activated according to the presentinvention and are included in the present invention are known to bepathogenic to human and/or other living organisms, for example, Ashbyagossypii (Ashby et Nowell) Guillermond, ACCC2001, AS2.475, AS2.1176;Blastomyces dermatitidis Gilehrist et Stikes, ID(D 10)23; Candidaalbicans (Robin) Berkhout, ACCC2002, AS2.538, ID 16u(C1)u, ID 61v(C1)v;Candida parakrusei (Castellani et Chalmer) Langeron et Guerra, ID 19a(C4)a, ID 19 b(C4)b, ID 19 c(C4)c, ID 19 d(C4)d; Candida tropicalis(Castellani) Berkout, ID 17 a(C₂)a, ID 17 b(C₂)b, ID 17 d(C₂)d;Citeromyces matritensis (Santa Maria) Santa Maria, AS2.1401;Crebrothecium ashbyii (Guillermond) Routein, ACCC2013, ACCC2014;Cryptococcus laurentii (Kufferath) Skinner, ACCC2007, AS2.114, ID 95(y₂); Cryptococcua neoformans (Sanfelice) Vuillemin, ID 25 u(D₂)u, ID 25v(D₂)v, ID 25 w(D₂)vw, Debaryomyces hansenii (Zopf) Lodder et Kreger-vanRij, ACCC2010; Debaryomyces Kloeckeri Guilliermond et Peju, ACCC2008,ACCC2009; Debaryomyces sp., ACCC2011, ACCC2012; Endomycopsis fibuligera(Lindner) Dekker, ACCC2015, AS2.1145. Under certain circumstances, itmay be less preferable to use such pathogenic yeasts in the biologicalfertilizer of the invention, for example, if such use in an open fieldmay endanger the health of human and/or other living organisms.

Yeasts of the Saccharomyces genus are generally preferred. Among strainsof Saccharomyces cerevisiae, Saccharomyces cerevisiae Hansen is apreferred strain. The most preferred strains of yeast are Saccharomycescerevisiae Hansen strains having accession numbers AS2.501, AS2.535,AS2.441, AS2.406, AS2.382, and AS2.16 as deposited at the China GeneralMicrobiological Culture Collection Center (CGMCC). Generally, the yeaststrains can be obtained from private or public laboratory cultures, orpublically accessible culture deposits, such as the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209and the China General Microbiological Culture Collection Center (CGMCC),China Committee for Culture Collection of Microorganisms, Institute ofMicrobiology, Chinese Academy of Sciences, Haidian, P.O. Box 2714,Beijing, 100080, China.

Although it is preferred, the preparation of the yeast cell componentsof the invention is not limited to starting with a pure strain of yeastEach yeast cell component may be produced by culturing a mixture ofyeast cells of different species or strains. The constituents of a yeastcell component can be determined by standard yeast identificationtechniques well known in the art.

Some yeasts may perform one of the desired functions more efficientlythan others. The ability of any species or strain of yeast to performone of the six desired functions before or after culturing under theconditions of the invention can readily be tested by methods known inthe art. For example, the amount of nitrogen fixed can be determined bya modified acetylene reduction method as described in U.S. Pat. No5,578,486 which is incorporated herein by reference in its entirety. Themodified acetylene reduction method determines the amount of nitrogenfixed by measuring the decrease in molecular nitrogen in a volume ofair. The amount of nitrogen fixed can also be determined by measurementof the ammonia and nitrates produced by the yeast cells (see, forexample, Grewling et al., 1965, Cornell Agr Exp Sta Bull 960:22-25). Forthe other functions, the amount of phosphorus available to plants as aresult of conversion from insoluble or biologically-unavailablephosphorus compounds can be determined by the molybdenum blue method(see, for example, Murphy et al., 1962, Analytica Chimica Acta 27:31-36)or the UV absorption method; whereas the amount of available potassiumconverted from insoluble or biologically-unavailable potassium compoundscan be determined, for example, by flame atomic absorption spectroscopy(see, for example, Puchyr, et al., 1986, J. Assoc. Off. Anal. Chem.69:868-870). The ability of the yeasts to supply plant available N, P,and K after the biological fertilizer composition has been added to soilcan be tested by many techniques known in the art. For example,plant-available ammonia, nitrates, P, and K produced by the yeast cellsin soil can be extracted and quantitatively analyzed by the Morgan soiltest system (see, for example, Lunt et al., 1950, Conn Agr Exp Sta Bull541).

Without being bound by any theory or mechanism, the inventor believesthat the culture conditions activate and/or enhance the expression of agene or a set of genes in yeast such that the yeast cells become activeor more efficient in performing the respective functions.

According to the invention, the biological fertilizer compositionscomprises at least one yeast cell component capable of performing one ofthe following biological functions: (1) fixation of atmosphericnitrogen; (2) decomposition of insoluble or biologically-unavailablephosphorus minerals or compounds present in the fertilizer compositionor in soil; (3) decomposition of insoluble or biologically-unavailablepotassium minerals or compounds present in the fertilizer composition orin soil; (4) decomposition of complex or high molecular weight carbonmaterials or compounds present in the fertilizer composition or in soil;(5) production of excess growth factors in an amount that is sufficientto support the needs of other yeast strains in the fertilizercomposition; and (6) production of excess ATP in an amount that issufficient to support the needs of other yeast strains in the fertilizercomposition. In preferred embodiments, the biological fertilizercompositions can comprise from one yeast strain to up to six differentyeast species or strains, each cultured under specific conditions toinduce or maximize its ability to perform the respective functions. Itwill be understood that alternative formulations are also contemplated.Thus, if desired, the biological fertilizer composition may omit one ormore of the above-described yeast cell components. For example, in soilrich in biologically-available phosphorus, a fertilizer composition maybe formulated to lack the component consisting of phosphoruscompounds-decomposing yeast. In the most preferred embodiments of thepresent invention, a biological fertilizer composition that contains allsix yeast cell components as well as the organic and/or inorganicsubstrates is contemplated.

In another embodiment of the invention, where the yeast cells of thevarious yeast cell components are present in a mixture, the yeast cellscan be cultured under certain conditions such that the yeast cells withdifferent functions can supply each other with and/or rely on each otherfor nutrients and growth factors. As a result, a symbiosis-likerelationship is established among the various yeast cell components inthe fertilizer compositions of the invention. This culturing process isoptional but can improve the stability and efficiency of the biologicalfertilizer such that the fertilizer is made more suitable for long termuse in natural soil environments. The culturing conditions for thisoptional process are described in Section 5.7.

In yet another embodiment of the invention, the yeast cells may also becultured under certain conditions so as to adapt the yeast cells to aparticular type of soil. This culturing process is optional, and can beapplied to each yeast cell component separately or to a mixture of yeastcell components. The result is better growth and survival of the yeastsin a particular soil environment. The culturing conditions for thisoptional process are described in Section 5.8.

As used herein, the biological fertilizer composition supports orenhances plant growth, if in the presence of the biological fertilizerin the soil, or applied to the roots, stems, leaves or other parts ofthe plant, the plant or a part of the plant gains viability, size,weight, rate of germination, rate of growth, or rate of maturation.Thus, the biological fertilizer compositions have utility in any kind ofagricultural, horticultural, and forestry practices. The biologicalfertilizer compositions can be used for large scale commercial farming,in open fields or in greenhouse, or even in interiors for decorativeplants. Preferably, the biological fertilizer is used to enhance thegrowth of crop plants, such as but not limited to cereal crops,vegetable crops, fruit crops; flower crops, and grass crops. Forexample, the biological fertilizer may be used with wheat, barley, corn,soybean, rice, oat, potato, apple, orange, tomato, melon, cherry, lemon,lettuce, carrot, sugar cane, tobacco, cotton, etc.

The biological fertilizer compositions may be applied in the same manneras conventional fertilizers. As known to those skilled in the relevantart, many methods and appliances may be used. In one embodiment, culturebroths of the yeast strains of the present invention are applieddirectly to soil or plants. In another embodiment, dried powders of theyeast strains of the present invention are applied to soil or plants. Inyet another embodiment, mixtures of the yeast cell components andorganic and inorganic substrate components of the present invention areapplied to soil or plants. The biological fertilizer compositions may beapplied to soil, by spreaders, sprayers, and other mechanized meanswhich may be automated. The biological fertilizer compositions may beapplied directly to plants, for example, by soaking seeds and/or roots,or spraying onto leaves. Such application may be made periodically, suchas once per year, or per growing season, or more frequently as desired.The biological fertilizer compositions of the invention can also be usedin conjunction or in rotation with other types of fertilizers.

Described respectively in Sections 5.1-5.6 are the yeast cell componentsused for nitrogen fixation, phosphorus compound decomposition, potassiumcompound decomposition, complex carbon compound decomposition, growthfactors production, and ATP production. Methods for preparing each yeastcell components are described. Section 5.7 describes the methods forestablishing a symbiosis-like relationship among yeast strains in afertilizer composition of the invention. Section 5.8 describes methodsfor adapting yeast cells of the invention to a particular type of soil.Section 5.9 describes the manufacture of the biological fertilizercompositions. Methods for the preparation of organic and inorganic rawmaterials and for the manufacture of the biological fertilizer,including mixing, drying, cooling, and packing, are also described. Invarious embodiments of the invention, standard techniques for handling,transferring, and storing microorganisms are used. Although it is notnecessary, sterile conditions or clean environments are desirable whencarrying out the processes of the invention.

5.1. Nitrogen-Fixing Yeast Cell Component

Nitrogen fixation is a process whereby atmospheric nitrogen is convertedinto ammonia and nitrates. Close to 800 species of naturally occurringmicroorganisms, mostly bacteria and cyanobacteria, from more than 70genera have been found to be able to fix nitrogen. Some of thenitrogen-fixing microorganisms, such as Rhizoboum, form symbioticassociation with plants, especially in the root of legumes. Others, suchas Azotobacter, are free-living and capable of fixing nitrogen in soil.

In the present invention, the ability of yeast to fix nitrogen isactivated or enhanced, and the resulting nitrogen-fixing yeast cells canbe used as a component of the biological fertilizer composition of theinvention.

According to the invention, yeast cells that have an enhanced ability tofix nitrogen are prepared by culturing the cells in the presence of anelectromagnetic field in an appropriate culture medium. The frequency ofthe electromagnetic field for activating or enhancing nitrogen fixitionin yeasts can generally be found within the range of 800 MHz-1000 MHz.After the yeast cells have been cultured for a sufficient period oftime, the cells can be tested for their ability to fix nitrogen bymethods well known in the art.

The method of the invention for making the nitrogen-fixing yeast cellsis carried out in a liquid medium. The medium contains sources ofnutrients assimilable by the yeast cells. In general, carbohydrates suchas sugars, for example, sucrose, glucose, fructose, dextrose, maltose,xylose, and the like and starches, can be used either alone or incombination as sources of assimilable carbon in the culture medium. Theexact quantity of the carbohydrate source or sources utilized in themedium depends in part upon the other ingredients of the medium but, ingeneral, the amount of carbohydrate usually varies between about 0.1%and 5% by weight of the medium and preferably between about 0.5% and 2%,and most preferably about 1%. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium.

Among the inorganic salts which can be incorporated in the culture mediaare the customary salts capable of yielding sodium, potassium, calcium,phosphate, sulfate, carbonate, and like ions. Non-limiting examples ofnutrient inorganic salts are CaCO₃, KH₂PO₄, MgSO₄, NaCl, and CaSO₄.TABLE I Composition for a culture medium for nitrogen-fixing yeastMedium Composition Quantity KH₂PO₄  0.2 g K₂HPO₄  0.2 g MgSO₄.7H₂O  0.25g CaCO₃.5H₂O  3.5 g CaSO₄.2H₂O  0.5 g NaCl  0.25 g Yeast extract paste 0.3 g Sucrose  12.0 g Distilled water or autoclaved water  1000 ml

It should be noted that the composition of the media provided in Table Iis not intended to be limiting. Various modifications of the culturemedium may be made by those skilled in the art, in view of practical andeconomic considerations, such as the scale of culture and local supplyof media components.

The process is initiated by inoculating each 100 ml of medium with 1 mlof an inoculum of the selected yeast strain(s) at a cell density of10²-10⁵ cell/ml, preferably 3×10²-10⁴ cell/ml. The process can be scaledup or down according to needs. The yeast culture is grown for about12-24 hours, preferably for about 24 hours, in the presence of anelectromagnetic field. The electromagnetic field, which can be appliedby any means known in the art, has a frequency in the range of 860 to870 MHz, preferably at about 865 MHz, more preferably in the range of865.522 to 865.622 MHz, and most preferably at 865.572 MHz. Theamplitude of the field is in the range of 1000-2000 mV, preferably atabout 1250 mV. After this first period of culture, the yeast cells arefurther incubated under substantially the same conditions forapproximately another 24 hours, except that the amplitude is increasedto a higher level in the range of 4000-5000 mV, preferably to about 4656mV. An exemplary set-up of the culture process is depicted in FIG. 1.The process of the invention is carried out at temperatures ranging-fromabout 25° to 30° C.; however, it is preferable to conduct the process at28° C. The culturing process may preferably be conducted underconditions in which the concentration of dissolved oxygen is between0.025 to 0.8 mol/m³, preferably 0.4 mol/m³. The oxygen level can becontrolled by any conventional means known to one skilled in the art,including but not limited to stirring and/or bubbling.

At the end of the culturing process, the nitrogen-fixing yeast cells maybe recovered from the culture by various methods known in the art, andstored at a temperature below about 0° C. to 4° C. The nitrogen-fixingyeast cells may also be dried and stored in powder form.

Any methods known in the art can be used to test the cultured yeastcells for their ability to fix nitrogen. For example, a modifiedacetylene reduction method for measuring nitrogen fixed bymicroorganisms is used to evaluate the nitrogen-fixing capability of theprepared yeast. The modified-acetylene reduction method is described inU.S. Pat. No.5,578,486 which is incorporated herein-by reference in itsentirety. For example, 1 ml of the prepared yeast culture is inoculatedinto 30 ml of a medium according to Table I in a sealed 250 ml flask.The culture is incubated at a temperature in the range of 20-28° C. for24-56 hours in the presence of air containing about 20% by volume oxygenand 80% by volume nitrogen. The amount of nitrogen fixed can then bedetermined by measuring the decrease in nitrogen from the air by anymeans-known in the art, such as but not limited to gas chromatography.The amount of nitrogen fixed by the yeast cells of the invention is atleast about 10 mg for each gram of yeast dry weight. For example, afteractivation, the amount of nitrogen fixed by Saccharomyces cerevisiaeHansen strain AS2.501, can reach about 11200 mg/g.

5.2. Phosphorus-Decomposing Yeast Cell Component

The phosphorus compound-decomposing (P-decomposing) yeast of theinvention converts insoluble or biologically-unavailablephosphorus-containing substances, such as rock phosphate, into solublephosphorous compounds so that they become available to plants.

In the present invention, the ability of yeast to decompose insolublephosphorus-containing substances is activated or enhanced, and theresulting P-decomposing yeast cells can be used as a component of thebiological fertilizer composition of the invention.

According to the invention, yeast cells that are capable ofP-decomposing are prepared by culturing the cells in the presence of anelectromagnetic field in an appropriate culture medium. The frequency ofthe electromagnetic field for activating or enhancing P-decomposition inyeasts can generally be found in the range of 300 MHz to 500 MHz. Afterthe cells have been cultured for a sufficient period of time, the cellscan be tested for their ability to decompose phosphorus-containingsubstances by methods well known in the art.

The method of the invention for making the P-decomposing yeast cells iscarried out in a liquid medium. The medium contains sources of nutrientsassimilable by the yeast cells. In general, carbohydrates such assugars, for example, sucrose, glucose, fructose, dextrose, maltose,xylose, and the like and starches, can be used either alone or incombination as sources of assimilable carbon in the culture medium. Theexact quantity of the carbohydrate source or sources utilized in themedium depends in part upon the other ingredients of the medium but, ingeneral, the amount of carbohydrate usually varies between about 0.1%and 5% by weight of the medium and preferably between about 0.5% and 2%,and most preferably about 1.5%. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium.

Among the inorganic salts which can be incorporated in the culture mediaare the customary salts capable of yielding sodium, potassium, calcium,sulfate, carbonate, and like ions. Non-limiting examples of nutrientinorganic salts are CaCO₃, MgSO₄, NaCl, and CaSO₄. Insolublephosphorus-containing substances in a suitable form are also included inthe media Non-limiting examples include powder of rock phosphate of ≧200mesh. Other insoluble phosphorus-containing substances can also be usedeither separately or in combination. TABLE II Composition for a culturemedium for P-decomposing yeast Medium Composition Quantity Sucrose   15g NaCl  1.2 g MgSO₄.7H₂O  0.2 g CaCO₃.5H₂O  3.0 g CaSO₄.2H₂O  0.3 g KNO₃ 0.3 g Yeast extract paste  0.5 g Rock phosphate  1.2 g; Powder of >200mesh Autoclaved water  1000 ml

It should be noted that the composition of the media provided in TableII is not intended to be limiting. Various modifications of the culturemedium may be made by those skilled in the art, in view of practical andeconomic considerations, such as the scale of culture and local supplyof media components.

The process is initiated by inoculating each 100 ml of medium with 1 mlof an inoculum of the selected yeast strain(s) at a cell density of10²-10⁵ cell/ml, preferably 3×10²-10⁴ cell/ml. The process can be scaledup or down according to needs. The yeast culture is grown for about12-24 hours, preferably for about 24-hours, in the presence of anelectromagnetic field. The electromagnetic field, which can be appliedby any means known in the art, has a frequency in the range of 360 to370 MHz, preferably at about 366 MHz, more preferably in the range of366.199 to 366.287 MHz, and most preferably at 366.243 MHz. Theamplitude of the field is in the range of 1000 to 2000 mV, preferably atabout 1230 mV. After this first period of culture, the yeast cells arefurther incubated under substantially the same conditions forapproximately another 24 hours, except that the amplitude is increasedto a higher level in the range of 4000 to 5000 mV, preferably to about4570 mV. An exemplary set-up of the culture process is depicted inFIG. 1. The process of the invention is carried out at temperaturesranging from about 25° to 30° C.; however, it is preferable to conductthe process at 28° C. The culturing process may preferably be conductedunder conditions in which the concentration of dissolved oxygen isbetween 0.025 to 0.8 mol/m³, preferably 0.4 mol/m³. The oxygen level canbe controlled by any conventional means known to one skilled in the art,including but not limited to stirring and/or bubbling.

At the end of the culturing process, the P-decomposing yeast cells maybe recovered from the culture by various methods known in the art, andstored at a temperature below about 0° C. to 4° C. The P-decomposingyeast cells may also be dried and stored in powder form.

Any methods known in the art can be used to test the cultured yeastcells for their ability to decompose insoluble phosphorus-containingsubstances. In one embodiment, 1 ml of the prepared yeast culture isinoculated into 30 ml of a medium according to Table II. The culture isincubated at a temperature in the range of 20-28° C. for 24-56 hours.The amount of biologically available phosphorus in the form of PO₄ ³⁻ inthe culture can then be determined by any methods known in the art,including but not limited to UV absorption spectroscopy. The amount ofPO₄ ³⁻ in the culture is increased by at least 10 mg for each gram ofyeast dry weight. For example, after activation, the amount of PO₄ ³⁻ ina culture of Saccharomyces cerevisiae Hansen strain AS2.535 is increasedto about 4460 mg/g.

5.3. Potassium-Decomposing Yeast Cell Component

The potassium compound-decomposing (K-decomposing) yeast of theinvention converts insoluble potassium-containing substances, such aspotassium mica, into soluble potassium so that they become available toplants.

In the present invention, the ability of a plurality of yeast cells todecompose insoluble potassium-containing substances is activated orenhanced, and the resulting K-decomposing yeast cells can be used as acomponent of the biological fertilizer composition of the invention.

According to the present invention, yeast cells that are capable ofK-decomposing are prepared by culturing the cells in the presence of anelectromagnetic field in an appropriate culture medium. The frequency ofthe-electromagnetic field for activating or enhancing K-decomposition inyeasts can generally be found in the range of 100 MHz-300 MHz After theyeast cells have been cultured for a sufficient period of time, thecells can be tested for their ability to decompose potassium-containingsubstances by methods well known in the art.

The method of the invention for making the K-decomposing yeast cells iscarried out in a liquid medium. The medium contains sources of nutrientsassimilable by the yeast cells. In general, carbohydrates such assugars, for example, sucrose, glucose, fructose, dextrose, maltose,xylose, and the like and starches, can be used either alone or incombination as sources of assimilable carbon in the culture medium. Theexact quantity of the carbohydrate source or sources utilized in themedium depends in part upon the other ingredients of the medium but, ingeneral, the amount of carbohydrate usually varies between about 0.1%and 5% by weight of the medium and preferably between about 0.5% and 2%,and most preferably about 1.5%. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium.

Among the inorganic salts which can be incorporated in the culture mediaare the customary salts capable of yielding sodium, calcium, phosphate,sulfate, carbonate, and like ions. Non-limiting examples of nutrientinorganic salts are (NH₄)₂HPO₄, CaCO₃, MgSO₄, NaCl, and CaSO₄. Insolublepotassium-containing substances in a suitable form are also included inthe media. Non-limiting examples include powder of potassium mica of≧200 mesh. Other insoluble potassium-containing substances can also beused either separately or combined. TABLE III Composition for a culturemedium for K-decomposing yeast Medium Composition Quantity Sucrose   15g NaCl  1.2 g MgSO₄.7H₂O  0.2 g CaCO₃.5H₂O  3.0 g CaSO₄.2H₂O  0.3 g(NH₄)₂HPO₄  0.3 g Yeast extract paste  0.3 g Potassium mica  1.2 g,Powder of >200 mesh Autoclaved water  1000 ml

It should be noted that the composition of the media provided in TableIII is not intended to be limiting. Various modifications of the culturemedium may be made by those skilled in the art, in view of practical andeconomic considerations, such as the scale of culture and local supplyof media components.

The process is initiated by inoculating each 100 ml of medium with 1 mlof an inoculum of the selected yeast stain(s) at a cell density of10²-10⁵ cell/ml, preferably 3×10²-10⁴ cell/ml. The process can be scaledup or down according to needs. The yeast culture is grown for about12-24 hours, preferably for about 24 hours, in the presence of anelectromagnetic field. The electromagnetic field, which can be appliedby any means known in the art, has a frequency in the range of 250-260MHz, preferably at about 255 MHz, more preferably in the range of255.388 to 255.462 MHz, and most preferably at 255.425 MHz. Theamplitude of the field is in the range of 1000-2000 mV, preferably atAbout 1340 mV. After this first period of culture, the yeast cells arefurther incubated under substantially the same conditions forapproximately another 24 hours, except that the amplitude is increasedto a higher level in the range of 4000-5000 mV, preferably to about 4850mV. An exemplary set-up of the culture process is depicted in FIG. 1.The process of the invention is carried out at temperatures ranging fromabout 25° to 30° C.; however, it is preferable to conduct the process at28° C. The culturing process may preferably be conducted underconditions in which the concentration of dissolved oxygen is between0.025 to 0.8 mol/m³, preferably 0.4 mol/m³. The oxygen level can becontrolled by any conventional means known to one skilled in the art,including but not limited to stirring and/or bubbling.

At the end of the culturing process, the K-decomposing yeast cells maybe recovered from the culture by various methods known in the art, andstored at a temperature below about 0-4° C. The K-decomposing yeastcells may also be dried and stored in powder form.

Any methods known in the art can be used to test the cultured yeastcells for their ability to decompose insoluble potassium-containingsubstances. In one embodiment, 1 ml of the prepared yeast culture isinoculated into 30 ml of a medium according to Table III. The culture isincubated at a temperature in the range of 20-28° C. for 24-56 hours.The amount of biologically available potassium in the form of K⁺ in theculture can then be determined by any methods known in the art,including but not limited to atomic absorption spectrometry. The amountof K⁺ in the culture is increased by at least 10 mg for each gram ofyeast dry weight. For example, after activation, the amount of K⁺ in aculture of Saccharomyces cerevisiae Hansen strain AS2.441 can reachabout 4050 mg/g.

5.4. Complex Carbon-Decomposing Yeast Cell Component

The carbon-decomposing (C-decomposing) yeast of the invention convertscomplex, usually high molecular weight, carbon compounds and material,such as cellulose, into simple carbohydrates, such as pentoses andhexoses. Such simple carbohydrates are utilized by other yeast cells tosupport their growth and activities.

In the present invention, the ability of yeast to decompose complexcarbon compounds very efficiently is activated or enhanced, and theresulting C-decomposing yeast cells can be used as a component of thebiological fertilizer composition of the invention.

According to the present invention, yeast cells that are capable ofC-decomposition are prepared by culturing the cells in the presence ofan electromagnetic field in an appropriate culture medium. The frequencyof the electromagnetic field for C-decomposition in yeasts can generallybe found in the range of 1000 MHz-1200 MHz After the yeast cells havebeen cultured for a sufficient period of time, the cells can be testedfor their ability to decompose complex carbon compounds by methods wellknown in the art.

The method of the invention for making the C-decomposing yeast cells iscarried out in a liquid medium. The medium contains sources of nutrientsassimilable by the yeast cells. Complex carbon-containing substancessuch as cellulose, coal, etc., in a suitable form can be used as sourcesof carbon in the culture medium. The exact quantity of the carbon sourceor sources utilized in the medium depends in part upon the otheringredients of the medium but, in general, the amount of carbohydrateusually varies between about 0.1% and 5% by weight of the-medium andpreferably between about 0.1% and 1%, and most preferably about 0.5%.These carbon sources can be used individually, or several such carbonsources may be combined in the medium.

Among the inorganic salts which can be incorporated in the culture mediaare the customary salts capable of yielding sodium, calcium, phosphate,sulfate, carbonate, and like ions. Non-limiting examples of nutrientinorganic salts are (NH₄)₂HPO₄, CaCO₃, MgSO₄, NaCl, and CaSO₄. TABLE IVComposition for a culture medium for C-decomposing yeast MediumComposition Quantity Cellulose  5.0 g; Powder of >100 mesh NaCl  0.6 gMgSO₄.7H₂O  0.3 g CaCO₃.5H₂O  1.5 g CaSO₄.2H₂O  0.4 g (NH₄)₂HPO₄  0.3 gYeast extract paste  0.5 g K₂HPO₄  0.5 g Autoclaved water  1000 ml

It should be noted that the composition of the media provided in TableIV is not intended to be limiting. Various modifications of the culturemedium may be made by those skilled in the art, in view of practical andeconomic considerations, such as the scale of culture and local supplyof media components.

The process is initiated by inoculating each 100 ml of medium with 1 mlof an inoculum of the selected yeast strain(s) at a cell density of10²-10⁵ cell/ml, preferably 3×10²-10⁴ cell/ml. The process can be scaledup or down according to needs. The yeast culture is grown for about12-24 hours, preferably for about 24 hours, in the presence of anelectromagnetic field. The electromagnetic field, which can be appliedby any means known in the art, has a frequency in the range of 1087-1097MHz, preferably about at 1092, more preferably in the range of 1092.346to 1092.428 MHz, and most preferably at 1092.387 MHz. The amplitude usedcan be in the range of 1000-200 mV, preferably at about 1530 mV. Afterthis first period of culture, the yeast cells are further incubatedunder substantially the same conditions for approximately another 24hours, except that the amplitude is increased to a higher level in therange of 4000-5000 mV, preferably to about 4720 mV. An exemplary set-upof the culture process is depicted in FIG. 1. The process of theinvention is carried out at temperatures ranging from about 25° to 30°C.; however, it is preferable to conduct the process at 28° C. Theculturing process may preferably be conducted under conditions in whichthe concentration of dissolved oxygen is between 0.025 to 0.8 mol/m³,preferably 0.4 mol/m³. The oxygen level can be controlled by anyconventional means known to one skilled in the art, including but notlimited to stirring and/or bubbling.

At the end of the culturing process, the C-decomposing yeast cells maybe recovered from the culture by various methods known in the art, andstored at a temperature below about 0-4° C. The C-decomposing yeastcells may also be dried and stored in powder form.

Any methods known in the art can be used to test the cultured yeastcells for their ability to decompose complex-carbon containingsubstances. In one embodiment, 1 ml of the prepared yeast culture isinoculated into 30 ml of a medium according to Table IV. The culture isincubated at a temperature in the range of 20-28° C. for 24-56 hours.The amount of simple carbohydrates in the culture can then be determinedby any methods known in the art, including but not limited tochromatography and molecular fluorescence spectroscopy. Preferably, theamount of simple carbohydrates in the culture is increased by at least10 mg for each gram of yeast dry weight For example, after activation,the amount of simple carbohydrates in a culture of Saccharomycescerevisiae Hansen AS2.406 can reach 27200 mg/g.

5.5. Growth Factors Producing Yeast Cell Component

The growth factors producing (GP-producing) yeast of the presentinvention produces vitamins and other nutrients, such as but not limitedto, vitamin B-1, riboflavin (vitamin B-2), vitamin B-12, niacin (B-3),pyridoxine (B-6), pantothenic acid (B-5), folic acid, biotin,para-aminobenzoic acid, choline, inositol, in such amounts that cansupport the growth of other yeast strains. Such growth factors areproduced by yeast during the fermentation process.

In the present invention, the ability of yeast to overproduce growthfactors is activated or enhanced, and the resulting GP-producing yeastcells can be used as a component of the biological fertilizercomposition of the invention.

According to the present invention, yeast cells that are capable ofGP-producing are prepared by culturing the cells in the presence of anelectromagnetic field in an appropriate culture medium. The frequency ofthe electromagnetic field for activating or enhancing GP-production inyeasts can generally be found in the range of 1300 MHz-1500 MHz. Afterthe yeast cells have been cultured for a sufficient period of time, thecells can be tested for their ability to produce growth factors bymethods well known in the art.

The method of the invention for making the GP-producing yeast cells iscarried out in a liquid medium. The medium contains sources of nutrientsassimilable by the yeast cells. In general, carbohydrates such assugars, for example, sucrose, glucose, fructose, dextrose, maltose,xylose, and the like and starches, can be used either alone or incombination as sources of assimilable carbon in the culture medium. Theexact quantity of the carbohydrate source or sources utilized in themedium depends in part upon the other ingredients of the medium but, ingeneral, the amount of carbohydrate usually varies between about 0.1%and 5% by weight of the medium and preferably between about 0.5% and 2%,and most preferably about 0.8%. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium.

Among the inorganic salts which can be incorporated in the culture mediaare the customary salts capable of yielding sodium, calcium, phosphate,sulfate, carbonate, and like ions. Non-limiting examples of nutrientinorganic salts are NH₄NO₃, K₂HPO₄, CaCO₃, MgSO₄, NaCl, and CaSO₄. TABLEV Composition for a culture medium for GP-producing yeast MediumComposition Quantity Starch  8.0 g; Powder of >120 mesh NaCl  0.3 gMgSO₄.7H₂O  0.2 g CaCO₃.5H₂O  0.5 g CaSO₄.2H₂O  0.2 g NH₄NO₃  0.3 gK₂HPO₄  0.8 g Autoclaved water  1000 ml

It should be noted that the composition of the media provided in Table Vis not intended to be limiting. Various modifications of the culturemedium may be made by those skilled in the art, in view of practical andeconomic considerations, such as the scale of culture and local supplyof media components.

The process is initiated by inoculating each 100 ml of medium with 1 mlof an inoculum of the selected yeast strain(s) at a cell density of10²-10⁵ cell/ml, preferably 3'10²-10⁴ cell/ml. The process can be scaledup or down according to needs. The yeast culture is grown for about12-24 hours, preferably for about 24 hours, in the presence of anelectromagnetic field. The electromagnetic field, which can be appliedby any means known in the art, has a frequency in the range of 1382-1392MHz, preferably at about 1387 MHz, more preferably in the range of1387.517 to 1387.595 MHz, and most preferably at 1387.556 MHz. Theamplitude used can be in the range of 1000-2000 mV, preferably at about1620 mV. After this first period of culture, the yeast cells are furtherincubated under substantially the same conditions for approximatelyanother 24 hours, except that the amplitude is increased to a higherlevel in the range of 4000-5000 mV, preferably to about 4830 mV. Anexemplary set-up of the culture process is depicted in FIG. 1. Theprocess of the invention is carried out at temperatures ranging fromabout 25° to 30° C.; however, it is preferable to conduct the process at28° C. The culturing process may preferably be conducted underconditions in which the concentration of dissolved oxygen is between0.025 to 0.8 mol/m³, preferably 0.4 mol/m³. The oxygen level can becontrolled by any conventional means known to one skilled in the art,including but not limited to stirring and/or bubbling.

At the end of the culturing process, the GP-producing yeast cells may berecovered from the culture by various methods known in the art, andstored at a temperature below about 0-4° C. The GP-producing yeast cellsmay also be dried and stored in powder form.

Any methods known in the art can be used to test the cultured yeastcells for their ability to overproduce growth factors. In oneembodiment, 1 ml of the prepared yeast culture is inoculated into 30 mlof a medium according to Table V. The culture is incubated at atemperature in the range of 20-28° C. for 32-48 hours. The amount ofgrowth factors as represented by the total amount of vitamin B1, B2, B6,and B12 in the culture can then be determined by any methods known inthe art, including but not limited to high performance liquidchromatography (HPLC). The amount of growth factors in the culture isincreased by at least 10 mg for each gram of yeast dry weight. Forexample, after activation, the amount of vitamin B1, B2, B6, and B12 ina culture of Saccharomyces cerevisiae Hansen AS2.382 can reach anaggregate of 6120 mg/g.

5.6. ATP-Producing Yeast Cell Component

The ATP-producing yeast of the present invention is capable ofoverproducing ATP in such amounts that can support the growth of otheryeast strains in the biological fertilizer composition.

In the present invention, the ability of yeast to overproduce ATP isactivated or enhanced, and the resulting ATP-producing yeast cells canbe used as a component of the biological fertilizer composition of theinvention.

According to the present invention, yeast cells that are capable ofenhanced ATP-production are prepared by culturing the cells in thepresence of an electric field in an appropriate culture medium. Thefrequency of the electromagnetic field for activating or enhancingATP-production in yeasts can generally be found in the range of 1600MHz-1800 MHz. After sufficient time is given for the cells to grow, thecells can be tested for their enhanced ability to produce ATP by methodswell known in the art.

The method of the invention for making the ATP-producing yeast cells iscarried out in a liquid medium. The medium contains sources of nutrientsassimilable by the yeast cells. In general, carbohydrates such assugars, for example, sucrose, glucose, fructose, dextrose, maltose,xylose, and the like and starches, can be used either alone or incombination as sources of assimilable carbon in the culture medium. Theexact quantity of the carbohydrate source or sources utilized in themedium depends in part upon the other ingredients of the medium but, ingeneral, the amount of carbohydrate usually varies between about 0.1%and 5% by weight of the medium and preferably between about 0.5% and 2%,and most preferably about 0.8%. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium.

Among the inorganic salts which can be incorporated in the culture mediaare the customary salts capable of yielding sodium, calcium, phosphate,sulfate, carbonate, and like ions. Non-limiting examples of nutrientinorganic salts are (NH₄)₂HPO₄, CaCO₃, MgSO₄, NaCl, and CaSO₄. TABLE VIComposition for a culture medium for ATP-producing yeast MediumComposition Quantity Starch  10.0 g NaCl  0.2 g MgSO₄.7H₂O  0.2 gCaCO₃.5H₂O  0.8 g CaSO₄.2H₂O  0.2 g NH₄NO₃  0.2 g K₂HPO₄  0.5 gAutoclaved water  1000 ml

It should be noted that the composition of the media provided in TableVI is not intended to be limiting. Various modifications of the culturemedium may be made by those skilled in the art, in view of practical andeconomic considerations, such as the scale of culture and local supplyof media components.

The process is initiated by inoculating each 100 ml of medium with 1 mlof an inoculum of the selected yeast strain(s) at a cell density of10²-10⁵ cell/ml, preferably 3×10²-10⁴ cell/ml. The process can be scaledup or down according to needs. The yeast culture is grown for about12-24 hours, preferably for about 24 hours, in the presence of anelectromagnetic field. The electromagnetic field, which can be appliedby any means known in the art, has a frequency in the range of 1690-1700MHz, preferably at about 1694 MHz, more preferably in the range of1694.328 to 1694.402 MHz, and most preferably at 1694.365 MHz. Theamplitude of the field is in the range of 1000-2000 mV, preferably atabout 1470 mV. After this first period of culture, the yeast cells arefurther incubated under substantially the same conditions forapproximately another 24 hours, except that the amplitude is increasedto a higher level in the range of 4000-5000 mV, preferably to about 4780mV. An exemplary set-up of the culture process is depicted in FIG. 1.The process of the invention is carried out at temperatures ranging fromabout 25° to 30° C.; however, it is preferable to conduct the process at28° C. The culturing process may preferably be conducted underconditions in which the concentration of dissolved oxygen is between0.025 to 0.8 mol/m³, preferably 0.4 mol/m³. The oxygen level can becontrolled by any conventional means known to one skilled in the art,including but not limited to stirring and/or bubbling.

At the end of the culturing process, the ATP-producing yeast cells maybe recovered from the culture by various methods known in the art, andstored at a temperature below about 0-4° C. The ATP-producing yeastcells may also be dried and stored in powder form.

Any methods known in the art can be used to test the cultured yeastcells for their ability to overproduce ATP. In one embodiment, 1 ml ofthe prepared yeast culture is inoculated into 30 ml of a mediumaccording to Table VI. The culture is incubated at a temperature in therange of 20-28° C. for 36-56 hours. The amount of ATP in the culture canthen be determined by any methods known in the art, including but notlimited to HPLC. The amount of ATP produced is increased by at leastabout 10 mg for each gram of yeast dry weight. For example, afteractivation, the amount of ATP produced in a culture of Saccharomycescerevisiae Hansen strain AS2.16 can reach about 3320 mg/g.

5.7. Formation of Symbiosis-Like Relationships

In another embodimemt of the present invention, yeast strains with thenewly activated or enhanced ability to fix nitrogen, decomposephosphorus-containing minerals or compounds, decompose insolublepotassium-containing minerals or compounds, and decompose complex carbonmaterials as described in Sections 5.1-5.4 are combined and cultured sothat they form a symbiosis-like relationship whereby they can growtogether without substantially relying on outside supplies of biologicalavailable nitrogen, phosphorus, potassium, and carbon nutrients. Thenutrients needed for growth are supplied by the respectivenutrient-producing yeast strain within the fertilizer composition byconverting biologically-unavailable nutrients from various sources intoavailable nutrients. The activity of each of the yeast strains inproducing the respective types of nutrient relates in part to the needsof other yeast cells as well as the plants. As a result, soluble,biologically-available nutrients will be converted when needed, therebyavoiding excess losses due to, for example, leaching.

The optional process which can be used to improve the performance of thebiological fertilizer is described as follows. Four strains of yeastsprepared according to Sections 5.1-5.4 are mixed and cultured in thepresence of an electromagnetic field in an appropriate liquid medium.The medium contains nitrogen, phosphorus, potassium, and carbonnutrients in biologically unavailable forms. As non-limiting examples,atomospheric nitrogen is used as the source of nitrogen nutrient, powderof phosphate rock is used as the source of phosphorus nutrient, powderof potassium mica is used as the source of potassium nutrient, andpowdered cellulose is used as the source of complex carbon nutrient.Other forms of insoluble phosphorus- and potassium-containing substancesand complex carbon compounds may also be used in place of or incombination with any of the above-identified minerals as sources ofphosphorus, potassium, and carbon nutrients. Among the inorganic saltswhich can be incorporated in the culture media are the customary saltscapable of yielding sodium, calcium, sulfate, carbonate, and like ions.Non-limiting examples of nutrient inorganic salts are CaCO₃, MgSO₄,NaCl, and CaSO₄. TABLE VII Composition for a culture medium forformation of symbiosis-like relation Medium Composition Quantity NaCl 0.5 g MgSO₄.7H₂O  0.4 g CaCO₃.5H₂O  3.0 g CaSO₄.2H₂O  0.3 g Yeastextract paste  0.3 g Potassium mica  1.2 g; Powder of >200 mesh Rockphosphate  1.2 g; Powder of >200 mesh Cellulose  5.0 g; Powder of >200mesh Autoclaved water  1000 ml

It should be noted that the composition of the media provided in TableVII is not intended to be limiting. Various modifications of the culturemedium may be made by those skilled in the art, in view of practical andeconomic considerations, such as the scale of culture and local supplyof media components.

The culturing process may preferably be conducted under conditions inwhich the concentration of dissolved oxygen is between 0.025 to 0.8mol/m³, preferably 0.4 mol/m³. The oxygen level can be controlled by anyconventional means known to one skilled in the art, including but notlimited to stirring-and/or bubbling. The process of the invention iscarried out at temperatures ranging from about 25° to 30° C.; however,it is preferable to conduct the process at 28° C. The process isinitiated in sterilized medium by inoculating typically about 20 ml ofeach inoculum of the four strains of yeast cells, each at a cell densityof about 10⁸ cell/ml. The optional process can be scaled up or downaccording to needs.

The yeast culture is grown for 12-72 hours, preferably for about 48hours, in the presence of four independent electromagnetic fields. Theelectromagnetic fields, which can be applied by a variety of means, eachhas the following respective frequencies: (1) in the range of 860 to 870MHz, preferably at about 865 MHz, more preferably in the range of865.522 to 865.622 MHz, and most preferably at 865.572 MHz, fornitrogen-fixing; (2) in the range of 360-370 MHz, preferably at about366 MHz, more preferably in the range of 366.199 to 366.287 MHz, andmost preferably at 366.243 MHz, for phosphorus-decomposing; (3) in therange of 250-260 MHz, preferably at about 255 MHz, more preferably inthe range of 255.388 to 255.462 MHz, and most preferably at 255.425 MHz,for potassium-decomposing; and (4) in the range of 1087-1097 MHz,preferably about at 1092, more preferably in the range of 1092.346 to1092.428 MHz, and most preferably at 1092.387 MHz, for complexcarbon-decomposing. The amplitude of each electromagnetic field isrepeatedly cycled between 0-3000 mV, preferably between 20-1800 mV, insteps of 1 mV at a rate of 18-23 minutes per complete cycle. Anexemplary set-up of the culture process is depicted in FIG. 2.

5.8. Soil Adaptation

The yeast strains of the invention must also be able to grow and performtheir respective functions in various types of soils. The ability of theyeast strains to survive and grow can be enhanced by adapting the yeaststrains of the invention to a particular soil condition.

In another embodiment of the invention, yeast cells prepared accordingto any one of Sections 5.1-5.6 can be cultured separately or in amixture in a solid or semi-solid medium containing soil from one or moresoil sources. This optional process which can be used to improve theperformance of the biological fertilizer is described by way of anexample as follows.

A suspension containing 10 ml of yeasts at a density of 10⁶ cell/ml ismixed with a 1000 cm³ of the soil medium. The process can be scaled upor down according to needs. The mixture of yeast and soil is culturedfor about 48-96 hours, preferably for about 48 hours, in the presence ofan electromagnetic field. The electromagnetic field, which can beapplied by a variety of means, has a frequency that, depending on thestrain of yeast, corresponds to one of the frequencies described inSections 5.1-5.6. A field amplitude in the range of 100-3000 mV,preferably 2100 mV, can be used. The culture is incubated attemperatures that cycle between about 3° C. to about 48° C. For example,in a typical cycle, the temperature of the culture may start at 35-48°C. and be kept at this temperature for about 1-2 hours, then adjusted upto 42-45° C. and kept at this temperature for 1-2 hours, then adjustedto 26-30° C. and kept at this temperature for about 2-4 hours, and thenbrought down to 5-10° C. and kept at this temperature for about 1-2hours, and then the temperature may be raised again to 35-45° C. foranother cycle. The cycles are repeated until the process is completed.After the last temperature cycle is completed, the temperature of theculture is lowered to 3-4° C. and kept at this temperature for about 5-6hours. After adaptation, the yeast cells may be isolated and recoveredfrom the medium by conventional methods, such as filtration. The adaptedyeast cells can be stored under 4° C. An exemplary set-up of the cultureprocess is depicted in FIG. 3.

5.9. Separation or Enrichment of Yeast Cells

Yeast cells that have been adapted to form a symbiosis-like relationshipaccording to Section 5.7. can be separated or enriched in such a waythat each strain of yeast cells keep their acquired or enhancedfunctions. Separation of yeast cells is carried out according to methodsdescribed in U.S. Pat. No. 5,578,486 and Chinese patent publication CN1110317A which are incorporated herein by reference in its entirety. Thefrequency used for activating the yeast cells may be used during theseparation process. The separated yeast cells can then be dried, andstored.

5.10. Manufacture of the Biological Fertilizers

In addition to yeast cell components, various organic and inorganic rawmaterials can also be included in the biological fertilizer compositionsof the invention. The preparation of such materials as well as the stepsinvolved in the manufacture of the biological fertilizer are describedherein.

5.10.1. Preparation of the Organic and Inorganic Substrate Components

A wide range of organic and inorganic materials can be used in thebiological fertilizer compositions of the present invention. Organicmaterials, such as but not limited to coal-mine waste and weatheredcoal, or any materials that contain more than 20% of organic substances,can be used as sources of carbon to support the growth of plants andyeasts. Combinations and mixtures of such organic materials can also beused. Organic compounds present in such materials are decomposed by theyeast capable of breaking complex or high molecular weight carbon-chainmolecules into simple carbon compounds so that they can be used byplants and other yeast cells in the fertilizer.

Inorganic materials, such as but not limited to phosphate rock andpotassium mica, are included as sources of phosphorus and potassiumrespectively. Other phosphorous- or potassium-containing materials andminerals can also be used. These inorganic compounds are decomposed byK-decomposing and P-decomposing yeast cells into biologically availablepotassium and biologically available phosphorus that can be used by thegrowing plants as well as the yeast cells in the fertilizer. Any organicor inorganic material may be used alone or in combination or insubstitution with any other materials in the present invention.Alternatively, one or more organic or inorganic ingredients may beomitted, or substituted by another if it is deemed desirable by theparticular application. For example, potassium mica can be omitted ifthe soil contains sufficient potassium minerals.

The organic and inorganic materials used in the invention should notcontain amounts of toxic substances or microorganisms that can inhibitthe growth of the yeast cells or plants.

The organic and inorganic components in the present invention are groundinto suitable forms and sizes before incorporated into the fertilizer.Typically, the organic or inorganic material is conveyed into a crusherwhere it is broken up into pieces of ≦5 cm in diameter. Any conventionalcrusher or equivalent machines can be used for this purpose. The piecesare then transferred to a grinder by any conveying means and ground to apowder of ≧150 mesh. Any grinder that allows fine grinding can be usedfor this purpose. The powder is then conveyed to an appropriate storagetank for storage until use with other components of the fertilizer. Aschematic illustration of the grinding process is shown in FIGS. 4 and5.

5.10.2. Fermentation Process Using Growth Factor-Producing Yeast

In the present invention, the preparation of GP-producing yeast iscarried out in a fermentation process using as seed the activated yeaststrain as described in Section 5.5. A schematic of the fermentationprocess is illustrated in FIG. 6.

The fermentation medium is prepared according to a ratio of 2.5 litersof water per kilogram of starch. Clean water, preferably water free ofany microorganisms, is used to prepare the fermentation medium. Thefermentation is carried out at a temperature between 20-30° C.,preferably between 25-28° C., in a clean environment and in a spacewhere there are no strong sources of electromagnetic fields, such aspower lines and power generators. Any equipments that contact thefermentation broth, including reactors, pipelines, and stirrers, must bethroughly cleaned before each use. The fermentation process normallylasts about 60-72 hours, depending on the fermentation temperature. Atleast 90% of the fermentation substrate is fermented. Fermentation ispreferably conducted under semi-aerobic conditions or conditions inwhich the oxygen level is about 20-60% of the maximal soluble oxygenconcentration. The oxygen level can be controlled by any conventionalmeans known to one skilled in the art, including but not limited tostirring and/or bubbling. After fermentation, the cell counts shouldreach about 2×10¹⁰ cells/ml. The fermentation broth is kept at atemperature in the range of 15-28° C. and must be used within 24 hours.Alternatively, the GP-producing yeasts can be drained, dried and storedin powder form.

5.10.3. Fermentation Process Using ATP-Producing Yeast

In the present invention, the preparation of ATP-producing yeast iscarried out by a fermentation process using as seed the adapted yeaststrain as described in Section 5.6. A schematic of the fermentationprocess is illustrated in FIG. 6.

The fermentation medium is prepared according to a ratio of 2.5 litersof water per kilogram of starch. Clean water, preferably water free ofany microorganisms, most preferably autoclaved water, is used to preparethe fermentation media. The fermentation is carried out at a temperaturebetween 20-30° C., preferably between 25-28° C., in a clean environmentand in a space where there are no strong sources of electromagneticfields, such as power lines and power generators. Any equipments thatcontact the fermentation broth, including reactors, pipelines, andstirrers, must be throughly cleaned before each use. The fermentationprocess normally lasts about 60-72 hours, depending on the fermentationtemperature. At least 90% of the fermentation substrate is fermented.Fermentation is preferably conducted under semi-aerobic conditions orconditions in which the oxygen level is about 20-60% of the maximalsoluble oxygen-concentrtion. The oxygen level can be controlled by anyconventional means known to one skilled in the art, including but notlimited to stirring and/or bubbling. After fermentation, the cell countsshould reach about 2×10¹⁰ cells/ml. The fermentation broth is kept at atemperature in the range of 15-28° C. and must be used within 24 hours.Alternatively, the ATP-producing yeasts can be drained, dried and storedin powder form.

5.10.4. Preparation of Mixture of Raw Materials

Organic and inorganic raw materials are mixed in exemplary proportionsas shown in Table VIII. Appropriate amount of organic and inorganicmaterials prepared according to Section 5.10.1 and starch are conveyedto a mixer. Any conventional mixer, such as but not limited a rotarydrum mixer, can be used. The mixing tank is rotated constantly so thatpowders of inorganic material, organic material, and starch are mixedevenly. The mixture is then conveyed to a storage tank. The procedurefor mixing organic and inorganic substrate material is illustrated inFIG. 7. TABLE VIII Ratio of raw materials Material PercentageRequirement Powder of organic materials 60-71% ≧150 mesh, water content≦5% Powder of inorganic 15-20% ≧150 mesh, water content materials ≦3%Starch 10-15% regular starch powder, water content ≦8%

5.10.5. Preparation of Yeast Mixture

A yeast mixture is prepared in the exemplary proportions as shown inTable IX. Appropriate amounts of the six yeast strains in dried powderform prepared according to Section 5.1-5.6 are conveyed to a mixingtank. The yeasts are allowed to mix for about 10-20 minutes. The mixtureis then transferred to a storage tank. Any equipments used for mixingyeasts, including the mixing tank and the storage tank, must bethroughly cleaned, preferably sterilized, before each use. The yeastmixture is stored at a temperature below 20° C. and must be used within24 hours. The procedure for mixing yeasts is illustrated in FIG. 8.Alternatively, the mixture of six yeasts can be dried and stored inpowder form. TABLE IX Ratio of microorganisms Percentage Yeast Quantity(dry weight) Note Nitrogen-fixing yeast 1.0-2.0 kg 0.1-0.2% Dry yeastpowder Phosphorus-decomposing 1.0-2.0 kg 0.1-0.2% Dry yeast powder yeastPotassium-decomposing 1.0-2.0 kg 0.1-0.2% Dry yeast powder yeastCarbon-decomposing 1.0-2.0 kg 0.1-0.2% Dry yeast powder yeast Growthfactor-producing 25 L     1% Yeast yeast fermentation brothATP-producing yeast 75 L     3% Yeast fermentation broth

5.10.6. Manufacture of Biological Fertilizer

The biological fertilizer of the present invention is produced by mixingthe yeast mixture of Section 5.10.5 and the mixture of the organic andinorganic materials of Section 5.10.1 at a ratio according to Table X.For example, the yeasts and the organic and inorganic materials areconveyed to a granulizer to form granules. The granules of thefertilizer are then dried in a two-stage drying process. During thefirst drying stage, the fertilizer is dried in a first dryer at atemperature not exceeding 65° C. for a period of time not exceeding 10minutes so that yeast cells quickly become dormant. The fertilizer isthen send to a second dryer and dried at a temperature not exceeding 70°C. for a period of time not exceeding 30 minutes to further removewater. After the two stages, the water content should be lower than 5%.It is preferred that the temperatures and drying times be adhered to inboth drying stages so that yeast cells do not lose their vitality andfunctions. The fertilizer is then cooled to room temperature. Thefertilizer may also be screened in a separator so that fertilizergranules of a preferred size are selected. Any separator, such as butnot limited to a turbo separator with adjustable speed and screen sizes,can be used. The fertilizer of the selected size is then sent to a bulkbag filler for packing.

The production process is illustrated in FIGS. 9-11. FIG. 9 is aschematic illustration of the procedure for producing the fertilizerfrom its components. FIG. 10 is a schematic illustration of the dryingprocess. FIG. 11 is a schematic illustration of the cooling and packingprocess. TABLE X Composition of the biological fertilizer (for onemetric ton of fertilizer) Percentage (dry Quantity weight) Note Mixtureof raw materials 952-956 kg 95.2-95.4% Dry weight Mixture of yeasts 100L  4.4-4.8% Dry weight

6. EXAMPLE

The following example demonstrates the manufacture of a biologicalfertilizer composition of the present invention. This example is apreferred embodiment of the present invention.

Saccharomyces cerevisiae Hansen strains having accession numbersAS2.501, AS2.535, AS2.441, AS2.406, AS2.382, and AS2.16, each of whichis deposited in China General Microbiological Culture Collection Center(CGMCC), China Committee for Culture Collection of Microorganisms, wereused to prepare the yeast-cell components of the biological fertilizer.Yeast strain AS2.501 was cultured according to the method described inSection 5.1 for nitrogen-fixation. Yeast strain AS2.535 was culturedaccording to the method described in Section 5.2 for P-decomposition.Yeast strain AS2.441 was cultured according to the method described inSection 5.3 for K-decomposition. Yeast strain AS2.406 was culturedaccording to the method described in Section 5.4 for C-decomposition.Yeast strain AS2.382 was cultured according to the method described inSection 5.5 for growth factor-production. Yeast strain AS2.16 wascultured according to the method described in Section 5.6 forATP-production.

Coal mine waste and phosphate rock were used as organic and inorganicmaterials respectively. The coal mine waste used in the examplecontained at least 30% of organic substances. The phosphate rock used inthe example contained at least 25% of P₂O₅. Coal mine waste andphosphate rock were prepared according to Sections 5.10.1.

The production of growth factor-producing yeast was carried out in afermentation process using as seed the activated yeast strain AS2.382 asdescribed in Section 5.5. A schematic of the fermentation process isillustrated in FIG. 6. The fermentation medium was prepared according toa ratio of 2.5 liters of clean water per kilogram of starch. Thefermentation medium was inoculated according to a ratio of 10 ml of seedsolution per liter of medium. The fermentation was carried out at atemperature of 28±1° C. and an oxygen concentration of 0.4 mol/m³ in aclean environment where there were no sources of electromagnetic fieldsfor about 48 hours. After fermentation, the cell counts reached about2×10¹⁰ cells/ml.

The production of ATP-producing yeast was carried out in a fermentationprocess using as seed the activated yeast strain AS2.16 as described inSection 5.6. A schematic of the fermentation process is illustrated inFIG. 6. The fermentation medium was prepared according to a ratio of 2.5liters of clean water per kilogram, of starch. The fermentation mediumwas inoculated according to a ratio of 10 ml of seed solution per literof medium. The fermentation was carried out at a temperature of 28±1° C.and an oxygen concentration of 0.4 mol/m³ in a clean environment wherethere were no sources of electromagnetic fields for about 56 hours.After fermentation, the cell counts reached about 2×10¹⁰ cells/ml.

The mixture of raw materials was prepared according to Table XI and theprocedure in Section 5.10.4. TABLE XI Ratio of raw materials MaterialPercentage Requirement Powder of coal mine waste 65% ≧150 mesh, watercontent ≦5% Powder of phosphate rock 20% ≧150 mesh, water content ≦3%Starch 15% regular starch powder, water content ≦8%

The yeast mixture was prepared according to Table XII and the proceduredescribed in Section 5.10.5. TABLE XII Ratio of yeasts (for 1 metric tonof fertilizer) Percentage Yeast Quantity (dry weight) NoteNitrogen-fixing yeast 2.0 kg 0.2% Dry yeast powder AS2.502Phosphorus-decomposing 2.0 kg 0.2% Dry yeast powder yeast AS2.535Potassium-decomposing 2.0 kg 0.2% Dry yeast powder yeast AS2.441

1. A biological fertilizer composition comprising at least one of thefollowing yeast cell components: (a) a first yeast cell componentcomprising a first plurality of yeast cells that fix nitrogen; (b) asecond yeast cell component comprising a second plurality of yeast cellsthat decompose phosphorus compounds; or (c) a third yeast cell componentcomprising a third plurality of yeast cells that decompose potassiumcompounds.
 2. The biological composition of claim 1 further comprising:(d) a fourth yeast cell component comprising a fourth plurality of yeastcells that convert complex carbon compounds to simple carbohydrates; (e)a fifth yeast cell component comprising a fifth plurality of yeast cellsthat overproduce growth factors; and (f) a sixth yeast cell componentcomprising a sixth plurality of yeast cells that overproduce adenosinetriphosphate.
 3. A biological fertilizer composition comprising at leastone of the following yeast cell components: (a) a first yeast cellcomponent prepared by culturing a first plurality of yeast cells in afirst electromagnetic field having a frequency in the range of 860 to870 MHz and an amplitude in the range of 1000 to 5000 mV for a period oftime sufficient to cause said first plurality of yeast cells to fixnitrogen; (b) a second yeast cell component prepared by culturing asecond plurality of yeast cells in a second electromagnetic field havinga frequency in the range of 360 to 370 MHz and an amplitude in the rangeof 1000 to 5000 mV for a period of time sufficient to cause said secondplurality of yeast cells to decompose phosphorus compounds; or (c) athird yeast cell component prepared by culturing a third plurality ofyeast cells in a third electromagnetic field having a frequency in therange of 250 to 260 MHz and an amplitude in the range of 1000 to 5000 mVfor a period of time sufficient to cause said third plurality of yeastcells to decompose potassium compounds.
 4. The biological composition ofclaim 3 further comprising: (d) a fourth yeast cell component preparedby culturing a fourth plurality of yeast cells in a fourthelectromagnetic field having a frequency in the range of 1087 to 1097MHz and an amplitude in the range of 1000 to 5000 mV for a period oftime sufficient to cause said fourth plurality of yeast cells to convertcomplex carbon molecules to simple carbohydrates; (e) a fifth yeast cellcomponent prepared by culturing a fifth plurality of yeast cells in afifth electromagnetic field having a frequency in the range of 1382 to1392 MHz and an amplitude in the range of 1000 to 5000 mV for a periodof time sufficient to cause said fifth plurality of yeast cells tooverproduce growth factors; and (f) a sixth yeast cell componentprepared by culturing a sixth plurality of yeast cells in a sixthelectromagnetic field having a frequency in the range of 1690 to 1700MHz and an amplitude in the range of 1000 to 5000 mV for a period oftime sufficient to cause said plurality of yeast cells to overproduceadenosine triphosphate.
 5. The biological fertilizer composition ofclaim 2 or 4 further comprising an organic substrate component, aninorganic substrate component, or both organic and inorganic substratecomponents.
 6. The biological fertilizer composition of claim 2 or 4wherein each yeast cell component separately comprises yeast cells thatbelongs to a genus selected from the group consisting of Saccharomyces,Schizosaccharomyces, Sporobolomyces, Torulopsis, Trichosporon,Wickerhamia, Ashbya, Blastomyces, Candida, Citeromyces, Crebrothecium,Cryptococcus, Debaryomyces, Endomycopsis; Geotrichum, Hansenula,Kloeckera, Lipomyces, Pichia, Rhodosporidium, and Rhodotorula.
 7. Thebiological fertilizer composition of claim 2 or 4 wherein each yeastcell component comprises cells of a species of yeast selected from thegroup consisting of Saccharomyces cerevisiae, Saccharomyces chevalieri,Saccharomyces delbrueckii, Saccharomyces exiguus, Saccharomycesfermentati, Saccharomyces logos, Saccharomyces mellis, Saccharomycesmicroellipsoides, Saccharomyces oviformis, Saccharomyces rosei,Saccharomyces rouxii, Saccharomyces sake, Saccharomyces uvarum Beijer,Saccharomyces willianus, Saccharomyces sp., Saccharomyces ludwigii,Saccharomyces sinenses, Saccharomyces bailii, Saccharomycescarlsbergensis, Schizosaccharomyces octosporus, Schizosaccharomycespombe, Sporobolomyces roseus, Sporobolomyces salmonicolor, Torulopsiscandida, Torulopsis famta, Torulopsis globosa, Torulopsis inconspicua,Trichosporon behrendoo, Trichosporon capitatum, Trichosporon cutaneum,Wickerhamia fluoresens, Ashbya gossypii, Blastomyces dermatitidis,Candida albicans, Candida arborea, Candida guillermondii, CandidaKrusei, Candida lambica, Candida lipolytica, Candida parakrusei, Candidaparapsilosis, Candida parapsilosis, Candida pseudotropicalis, Candidapulcherrima, Candida robusta, Candida rugousa, Candida utilis,Citeromyces matritensis, Crebrothecium ashbyii, Cryptococcus laurentii,Cryptococcus neoformans, Debaryomyces hansenii, Debaryomyces kloeckeri,Endomycopsis fibuligera, Eremothecium ashbyii, Geotrichum candidum,Geotrichum ludwigii, Geotrichum robustum, Geotrichum suaveolens,Hansenula anomala, Hansenula arabitolgens, Hansenula jadinii, Hansenulasaturnus, Hansenula schneggii, Hansenula subpelliculosa, Kloeckeraapiculata, Lipomyces starkeyi, Pichia farinosa, Pichia membranaefaciens,Rhodosporidium toruloides, Rhodotorula aurantiaca, Rhodotorula glutinis,Rhodotorula minuta, Rhodotorula rubar, and Rhodotorula sinesis.
 8. Thebiological fertilizer composition of claim 2 or 4 wherein each yeastcell component comprises cells of Saccharomyces cerevisiae.
 9. Thebiological fertilizer composition of claim 2 or 4 wherein the yeastcells of each yeast cell component are separately cells of the yeaststrain Saccharomyces cerevisiae Hansen deposited at China GeneralMicrobiological Culture Collection Center having an accession numberselected from the group consisting of AS2.501, AS2.535, AS2.441,AS2.406, AS2.382, and AS2.16.
 10. The biological fertilizer compositionof claim 2 which comprises yeast cell components (a), (b), and (c) ofclaim
 1. 11. The biological fertilizer composition of claim 4 whichcomprises yeast cell components (a), (b), and (c) of claim
 3. 12. Thebiological fertilizer composition of claim 10 or 11 further comprisingan organic substrate component, an inorganic substrate component, orboth an organic and an inorganic substrate component.
 13. The biologicalfertilizer composition of claim 12 which comprises about 0.1 to 0.2% byweight of yeast cell component (a), about 0.1 to 0.2% by weight of yeastcell component (b), about 0.1 to 0.2% by weight of yeast cell component(c), about 0.1 to 0.2% by weight of yeast cell component (d), about 1%by weight of yeast cell component (e), about 3% by weight of yeast cellcomponent (f); about 65% by weight of organic substrate component; about19% by weight of inorganic substrate component; and about 14% by weightof starch. 14-20. (canceled)
 21. A biological fertilizer compositioncomprising (i) at least one of the following yeast cell components: (a)a first yeast cell component prepared by culturing a first plurality ofyeast cells in a first electromagnetic field having a frequency of about865.522 MHz and an amplitude of about 1250 mV for a period of 24 hoursand culturing said first plurality of yeast cells in the presence of asecond electromagnetic field having a frequency of about 865.522 MHz andan amplitude of about 4656 mV for a period of 24 hours so that saidfirst plurality of yeast cells can fix nitrogen; (b) a second yeast cellcomponent prepared by culturing a second plurality of yeast cells in afirst electromagnetic field having a frequency of about 366.243 MHz andan amplitude of about 1230 mV for a period of 24 hours and culturingsaid second plurality of yeast cells in the presence of a secondelectromagnetic field having a frequency of about 366.243 MHz and anamplitude of about 4570 mV for a period of 24 hours so that said secondplurality of yeast cells can decompose phosphorus compounds; or (c) athird yeast cell component prepared by culturing a third plurality ofyeast cells in a first electromagnetic field having a frequency of about255.425 MHz and an amplitude of about 1340 mV for a period of 24 hoursand culturing said third plurality of yeast cells in the presence of asecond electromagnetic field having a frequency of about 255.425 MHz andan amplitude of about 4850 mV for a period of 24 hours so that saidplurality of yeast cells can decompose potassium compounds; (ii) afourth yeast cell component prepared by culturing a fourth plurality ofyeast cells in a first electromagnetic field having a frequency of about1092.387 MHz and an amplitude of about 1530 mV for a period of 24 hoursand culturing said fourth plurality of yeast cells in the presence of asecond electromagnetic field having a frequency of about 1092.387 MHzand an amplitude of about 4720 mV for a period of 24 hours so that saidfourth plurality of yeast cells can convert complex carbon molecules tosimple carbohydrates; (iii) a fifth yeast cell component prepared byculturing a fifth plurality of yeast cells in a first electromagneticfield having a frequency of about 1387.556 MHz and an amplitude of about1620 mV for a period of 24 hours and culturing said fifth plurality ofyeast cells in the presence of a second electromagnetic field having afrequency of about 1387.556 MHz and an amplitude of about 4830 mV for aperiod of 24 hours so that said fifth plurality of yeast cells canoverproduce growth factors; and (iv) a sixth yeast cell componentprepared by culturing a sixth plurality of yeast cells in a firstelectromagnetic field having a frequency of about 1694.365 MHz and anamplitude of about 1470 mV for a period of 24 hours and culturing saidsixth plurality of yeast cells in the presence of a secondelectromagnetic field having a frequency of about 1694.365 MHz and anamplitude of about 4780 mV for a period of 24 hours so that saidplurality of yeast cells can overproduce adenosine triphosphate; whereinsaid yeast cell components comprise cells of Saccharomyces cerevisiae.22. The biological fertilizer composition of claim 21 wherein the firstyeast cell component comprises cells of the yeast strain Saccharomycescerevisiae Hansen AS2.501, the second yeast cell component comprisescells of the yeast strain Saccharomyces cerevisiae Hansen AS2.535, thethird yeast cell component comprises cells of the yeast strainSaccharomyces cerevisiae Hansen AS2.441, the fourth yeast cell componentcomprises cells of the yeast strain Saccharomyces cerevisiae HansenAS2.406, the fifth yeast cell component comprises cells of the yeaststrain Saccharomyces cerevisiae Hansen AS2.382, and the sixth yeast cellcomponent comprises cells of the yeast strain Saccharomyces cerevisiaeHansen AS2.16.
 23. The biological fertilizer composition of claim 21,wherein the pluralities of yeast cells are dried.
 24. A method ofactivating or enhancing the ability of a plurality of yeast cells to fixatmospheric nitrogen, comprising culturing said plurality of yeast cellsin the presence of an electromagnetic field having a frequency in therange of 850 to 860 MHz and an amplitude in the range of 1000 to 5000 mVfor a period of time sufficient to cause said plurality of yeast cellsto fix nitrogen.
 25. (canceled)
 26. A method of activating or enhancingthe ability of a plurality of yeast cells to decomposephosphorus-containing minerals or compounds, comprising culturing saidplurality of yeast cells in the presence of an electromagnetic fieldhaving a frequency in the range of 360 to 370 MHz and an amplitude inthe range of 1000 to 5000 mV for a period of time sufficient to causesaid plurality of yeast cells to decompose phosphorus compounds. 27.(canceled)
 28. A method of activating or enhancing the ability of aplurality of yeast cells to decompose potassium-containing minerals orcompounds, comprising culturing said plurality of yeast cells in thepresence of an electromagnetic field having a frequency in the range of250 to 260 MHz and an amplitude in the range of 1000 to 5000 mV for aperiod of time sufficient to cause said plurality of yeast cells todecompose potassium compounds.
 29. (canceled)
 30. A method of activatingor enhancing the ability of a plurality of yeast cells to decompose highmolecular weight carbon substances, comprising culturing said pluralityof yeast cells in the presence of an electromagnetic field having afrequency in the range of 1087 to 1097 MHz and an amplitude in the rangeof 1000 to 5000 mV for a period of time sufficient to cause saidplurality of yeast cells to convert complex carbon molecules to simplecarbohydrates.
 31. (canceled)
 32. A method of activating or enhancingthe ability of a plurality of yeast cells to overproduce growth factors,comprising culturing said plurality of yeast cells in the presence of anelectromagnetic field having a frequency in the range of 1382 to 1392MHz and an amplitude in the range of 1000 to 2000 mV for a period ofbetween 1000 to 5000 mV for a period of time sufficient to cause saidplurality of yeast cells to overproduce growth factors.
 33. The methodof claim 32, comprising culturing said plurality of yeast cells in thepresence of a first electromagnetic field having a frequency of about1387.556 and an amplitude of about 1620 mV for a period of 24 hours andculturing said fifth plurality of yeast cells in the presence of asecond electromagnetic field having a frequency of about 1387.556 MHzand an amplitude of about 4830 mV for a period of 24 hours so that saidfifth plurality of yeast cells can overproduce growth factors.
 34. Themethod of claim 32 or 33, further comprising inoculating a fermentationmedium comprising a starch solution at a concentration of about 400gram/liter with said yeast cells and allowing fermentation to proceed ata temperature between 20 to 30° C. until at least 90% of thefermentation substrate has been fermented.
 35. A method of activatingthe ability of a plurality of yeast cells to overproduce ATP, comprisingculturing said plurality of yeast cells in the presence of anelectromagnetic field having a frequency in the range of 1690 to 1700MHz and an amplitude in the range of 1000 to 5000 mV for a period oftime sufficient to cause said plurality of yeast cells to overproduceadenosine triphosphate.
 36. The method of claim 35, comprising culturingsaid plurality of yeast cells in the presence of a first electromagneticfield having a frequency of about 1694.365 MHz and an amplitude of about1470 mV for a period of 24 hours and culturing said sixth plurality ofyeast cells in the presence of a second electromagnetic field having afrequency of about 1694.365 MHz and an amplitude of about 4780 mV for aperiod of 24 hours so that said plurality of yeast cells can overproduceadenosine triphosphate.
 37. The method of claim 35 or 36, furthercomprising inoculating a fermentation medium comprising a starchsolution at a concentration of about 400 gram/liter with said yeastcells and allowing fermentation to proceed at a temperature between 20to 30° C. until at least 90% of the fermentation substrate has beenfermented.
 38. A method of forming a symbiosis-like relationship amongyeast cells components of a biological fertilzer, said method comprisesthe steps of: preparing a mixture comprising (a) a first yeast cellcomponent prepared by culturing a first plurality of yeast cells in afirst electromagnetic field having a frequency in the range of 860 to870 MHz and an amplitude in the range of 1000 to 5000 mV for a period oftime sufficient to cause said first plurality of yeast cells to fixnitrogen; (b) a second yeast cell component prepared by culturing asecond plurality of yeast cells in a second electromagnetic field havinga frequency in the range of 360 to 370 MHz and an amplitude in the rangeof 1000 to 5000 mV for a period of time sufficient to cause said secondplurality of yeast cells to decompose phosphorus compounds; (c) a thirdyeast cell component prepared by culturing a third plurality of yeastcells in a third electromagnetic field having a frequency in the rangeof 250 to 260 MHz and an amplitude in the range of 1000 to 5000 mV for aperiod of time sufficient to cause said third plurality of yeast cellsto decompose potassium compounds; and (d) a fourth yeast cell componentprepared by culturing a fourth plurality of yeast cells in a fourthelectromagnetic field having a frequency in the range of 1087 to 1097MHz and an amplitude in the range of 1000 to 5000 mV for a period oftime sufficient to cause said fourth plurality of yeast cells to convertcomplex carbon molecules to simple carbohydrates; and culturing saidmixture in the presence of an electromagnetic field having a pluralityof frequencies of 860 to 870 MHz, 360 to370 MHz, 250 to 260 MHz, and1087 to 1097 MHz and each frequency having an amplitude of 0 to 3000 mVfor a period of time sufficient to cause said yeast cell components toform symbiosis-like relationship, wherein the amplitude for eachfrequency in said electromagnetic field is cycled between 0 to 3000 mV.39. The method of claim 24, further comprising: preparing a mixturecomprising said plurality of yeast cells and soil; culturing saidmixture in an electromagnetic field having a frequency in the range of850 to 860 MHz and an amplitude in the range of 1000 to 5000 mV for aperiod of time sufficient to cause said plurality of yeast cells toadapt to soil.
 40. The method of claim 26, further comprising: preparinga mixture comprising said plurality of yeast cells and soil; culturingsaid mixture in an electromagnetic field having a frequency in the rangeof 360 to 370 MHz and an amplitude in the range of 1000 to 5000 mV for aperiod of time sufficient to cause said plurality of yeast cells toadapt to soil.
 41. The method of claim 28, further comprising: preparinga mixture comprising said plurality of yeast cells and soil; culturingsaid mixture in an electromagnetic field having a frequency in the rangeof 250 to 260 MHz and an amplitude in the range of 1000 to 5000 mV for aperiod of time sufficient to cause said plurality of yeast cells toadapt to soil.
 42. The method of claim 30, further comprising: preparinga mixture comprising said plurality of yeast cells and soil; culturingsaid mixture in an electromagnetic field having a frequency in the rangeof 1087 to 1097 MHz and an amplitude in the range of 1000 to 5000 mV fora period of time sufficient to cause said plurality of yeast cells toadapt to soil.
 43. The method of claim 32, further comprising: preparinga mixture comprising said plurality of yeast cells and soil; culturingsaid mixture in an electromagnetic field having a frequency in the rangeof 1382 to 1392 MHz and an amplitude in the range of 1000 to 5000 mV fora period of time sufficient to cause said plurality of yeast cells toadapt to soil.
 44. The method of claim 35, further comprising: preparinga mixture comprising said plurality of yeast cells and soil; culturingsaid mixture in an electromagnetic field having a frequency in the rangeof 1690 to 1700 MHz and an amplitude in the range of 1000 to 5000 mV fora period of time sufficient to cause said plurality of yeast cells toadapt to soil. 45-50. (canceled)
 51. A method for enhancing plant growthcomprising growing the plant in the presence of a biological fertilizercomposition of any one of the claims 1-13 and 21-23.